Sample exam in Molecular Biology

1. You have just isolated 0.03ng of DNA from a single human hair. You now want to amplify a single-copy nuclear gene. Do you have enough DNA for this? Explain your answer.
   

 

1. You are doing PCR using Drosophila template. If you wanted 1000 template molecules, how much DNA will you need? Note: The Drosophila genome is about 150Mb and your product is derived from a single copy sequence.
   

 

1. You want to make up 200ul of a PCR master mix that contains the following:
   

10U amplitaq gold    stock=5U/ul

1X buffer        stock=10X

2.0 mM Mg        stock=25mM

200uM dNTPs        stock=10mM of each dNTP

500uM each primer    stock=0.1nMoles/ul (20 mer)

10ng template        stock=5ug/50uls

Water to 200ul

 

How much of each ingredient do you add?

 

1. To save money, you want to order the least amount of primer possible. The company says the smallest amount you can order is 1 nM. How much master mix could you make from this? Note: Assume that your mix will contain 200uM primer and that the primer is 20 bases long (a 20 mer).
   

 

1. You want to make up 10 mls of a stock solution of 1M Tris (MW=121) and 0.1M EDTA (MW=360). The Tris is a solid and the EDTA comes from a 0.5M stock. How much of each ingredient would you need?
   

 

1. a. What is 'in situ' PCR?
   
   1. Why would you want to do it?
      
   2. How do you keep the in situ PCR product from floating away?
      

 

1. Explain what would happen to your sequencing traces if you did the following:
   
   1. Forgot to add the Exosap in the PCR product clean-up
      
   2. Forgot to add the EtOH to the sequencing reaction clean-up
      
   3. Forgot to denature the sample before loading on the gel.
      

 

1. What would happen to your PCR product (as seen on an agarose gel) if you did the following:
   
2. Misprogramed the thermocycler to do 60 rather than 30 cycles
   
3. Forgot to include the 10 min initial 94C denaturing step when using amplitaq gold
   
4. Added only one primer to the master mix rather than two.
   

 

1. What would happen to your DNA prep if you did the following:
   
2. Forgot to add the EtOH to the wash buffer
   
3. Centrifuged at full speed rather than 1/2 speed during the wash steps
   
4. Used 10mM Tris/1mM EDTA rather than water for the elution step
   

   
    

5. Your RT-PCR reaction didn't work! That is, when you run a gel you see no band of the expected size. You do see a smear below the 50bp marker and you see a very faint band 500 bp larger than your expected RT-PCR product. Outline the troubleshooting steps you would take to get the RT-PCR reaction to work.
   

   
    

6. a. Most thermocyclers have heated lids. Why?
   
   1. Would you ever not want a thermocycler with a heated lid? Explain your answer
      

 

1. Explain how dUTP and UNG can help prevent contamination in a PCR reaction. What types of contamination is not prevented by this procedure?
   

 

1. Explain the process of cycle sequencing with dRhodamine labeled dye terminators
   

 

1. When is it a good idea to sequence bulk PCR product and when is it a good idea to sequence cloned PCR products?
   

 

15. Describe two different types of probes used for real time PCR.

My solutions to Homework 2

Question:
Somewhere out on the Internet is a database of restriction enzymes.
a. Where is it located? What is the URL for the database file that could be used with the GCG software?
Answer: Database of restriction enzymes is located at REBASE.
The URL for the site is http://rebase.neb.com/rebase/rebase.html
The URL for the database file that could be used with the GCG software is http://rebase.neb.com/rebase/link_gcg

b. What does a typical entry look like for the restriction enzyme file that is formatted for use with the MacVector program?
Answer: Rebase Format #19 is used with the MacVector program.
Each entry is composed of lines. Different types of lines with their own formats are used to represent data. Each line begins with a two character line code which indicates the type of information provided in the line. “//” acts as the delimiter between individual entries.

Each entry in the database contains the following fields:
ID enzyme name
ET enzyme type
OS microorganism name
PT prototype
RS recognition sequence, cut site
MS methylation site (type)
CR commercial sources for the restriction enzyme
CM commercial sources for the methylase
RN [count]
RA authors
RL jour, vol, pages, year, etc.
//
Example of a typical entry:
ID M.BamHII
ET M
OS Bacillus amyloliquefaciens H
PT BamHII
RS GGATCC, ?;
MS 5;
RN [1]
RA Connaughton J.E., Vanek P.G., Chirikjian J.G.;
RL J. Cell Biol. 107:535a-535a(1988).
//

c. How is the database (formatted for MacVector) organized?
Answer: The database is organized in the form of a flatfile. It is a text only database with no graphics. It is in Bairoch format. It contains an alphabetical listing of types I, II and III restriction enzymes as well as methylases in a format that is compatible with a wide range of data banks (PROSITE, ENZYME, SwissProt, EMBL,ECD, EPD, HAEMB). Each entry is composed of lines. Different types of lines with their own formats are used to represent data. Each line begins with a two character line code which indicates the type of information provided in the line. “//” acts as the delimiter between individual entries.

1. What is the delimiter between individual restriction enzyme entries? How does the computer (or you) know when the information from one restriction enzyme stops and another one starts?
Answer: The delimiter between individual restriction enzyme entries is “//”.

2. Is this format similar to the format used by any other database? Which one?
Answer: I compared data in MacVector format with data in DNA Strider format in REBASE. Though similar in the fact that this format also provides information about restriction enzymes, and that data is organized in FASTA format, there are a few differences also, such as separation of fields etc, number of fields etc.

MacVector
DNA Strider
Each entry has many more descriptive fields than DNA Strider – enzyme name, enzyme type, organism name, prototype, recognition sequence, cut site, methylation site and commercial sources.
Each entry only has two descriptive fields – enzyme name, recognition sequence with cleavage site. Individual fields are separated by a comma (,)
Individual entry is separated by “//”
Individual entries start on a new line
Flatfile format
Flatfile format

Then I compared the MacVector format in REBASE database with the GenBank database: Though the format was similar in that both databases had a common delimiter “//”, most of the other attributes were very different.
MacVector
GenBank
Each entry starts with the ID field.
Each entry starts with the locus field.
Each field is represented by two characters line code such as ID, OS etc.
Each field is represented by one or more descriptive words such as definition, locus etc.
Information is only available in FASTA format.
Information is available in a wide range of formats such as FASTA, XML, Graphics etc.
Individual entries are separated by “//”
Individual entries are separated by “//”
It contains information about the restriction site of the enzyme and does not contain any information about the amino acid or nucleotide sequence
This database contains information about the nucleotide sequence. If coding for an expressed protein, it also contains the translated information.

Literature Search Questions
1) Select a protein and find the entries for this protein in the GenBank DNA database, the SwissProt database, and the PDB Protein database. List the attributes or features that are common to the databases and those which are unique to each.
Answer: I looked up the databases for β sub-unit of human follicle stimulating hormone.
PDB results:
URL: http://www.pdb.org/pdb/explore.do?structureId=1FL7
SwissProt results:
URL: http://au.expasy.org/uniprot/P01225
GenBank results:
URL: http://www.ncbi.nlm.nih.gov/entrez/viewer.fcgi?db=nuccore&id=EF198021

GenBank
SwissProt
PDB
The identifying code for the protein is the locus or the accession number which is usually an eight digit alphanumerical.
The identifying code is called the primary accession number. It is usually a six digit alphanumerical.
The unique identifying code for the protein is small, usually a four digit alphanumerical.
Genbank contains the following information in each entry: Locus, definition, accession #, version, keywords, source,organism, references, gene, mRNA and CDS.
SwissProt contains the following information in each entry: entry name, primary accession number, information about the name and origin of the protein, references, links to cross-references and the amino acid sequence.
PDB contains the following information in each entry: title, references, history, experimental information, molecular description and information about the structure of the protein.
GenBank gives the DNA sequence of the protein. It also contains the translated sequence of the sequence, if it is expressed.
SwissProt gives the amino acid sequence of the protein. Other sequence information can be found by following the links.
PDB gives detailed structural (3D) information about the protein with images and figures to help visualize the molecule. It also contains the amino acid sequence.
GenBank does not provide links for cross-referencing.
SwissProt has many links which allow for easy cross-referencing.
PDB also allows cross referencing via “external links”.
GenBank is a much larger database than SwissProt or PDB.
Swissprot is not as big a database as GenBank, but is bigger than PDB.
PDB is a relatively small database as many proteins that were available at SwissProt and GenBank could not be found here.
Information can be displayed in a wide array of formats such as FASTA, GenBank, XML, Graphical etc.
Sequence information is available only in two formats: SwissProt and FASTA. There is no graphical representation of data.
Sequence information is available in FASTA format. However, there is also ample graphical representation of the data.



2) How many secreted proteins have been discovered in humans? Explain what database you used, and what keywords you used to do the search.
Answer: I performed this search in SRS. I initially searched multiple databases, but the results were redundant, so I repeated the search using only one dataset, the patent proteins dataset, since results would not be duplicated here, and also because most secreted proteins would be entered here.
URL: http://srs.ebi.ac.uk/srsbin/cgi-bin/wgetz
Database searched: Patent Proteins
Search Field 1: All text - Secreted
Search Field 2: Organism name - human
Result: 10,319

Homework 2 Questions

Homework

Curious about how to answer these questions? Want to see an example of a homework answer? See this page for an example homework answer.
Database Questions
Somewhere out on the Internet is a database of restriction enzymes.
a. Where is it located? What is the URL for the database file that could be used with the GCG software?
b. What does a typical entry look like for the restriction enzyme file that is formatted for use with the MacVector program?
c. How is the database (formatted for MacVector) organized?
1. What is the delimiter between individual restriction enzyme entries? How does the computer (or you) know when the information from one restriction enzyme stops and another one starts?
2. Is this format similar to the format used by any other database? Which one?
Literature Search Questions
1) Select a protein and find the entries for this protein in the GenBank DNA database, the SwissProt database, and the PDB Protein database. List the attributes or features that are common to the databases and those which are unique to each.
2) How many secreted proteins have been discovered in humans? Explain what database you used, and what keywords you used to do the search.

Bioinformatics Workshop 3

Literature Workshop

In this workshop, we will be exploring how to search for sequence information using various web sites.
Web Resources
NCBI
Visit the NCBI website at http://www.ncbi.nlm.nih.gov
You have different options for searching for sequence information by querying the sequence annotation.
ENTREZ
PUBMED
OMIM
and several other databases
They are all linked together with links into the sequence databases.
When you do a search, you have to first ask yourself these questions
What information are you looking for?
What database would have that information?
Can you restrict your search to certain fields?
Try searching for literature about human growth hormone.
What MeSH term should you be using for that molecule?
What Database should you be searching?

Often, the best way to find something is to first, do some searches, assign some limits, then view the "history" and combine some queries. You can also do this using the Preview/Index option.

PDB
What is the unique identifying code for a protein structure of Lysozyme? You will find lots of lysozymes. Just pick one.
http://www.rcsb.org

SRS at EBI
http://srs.ebi.ac.uk
Search for Human Growth Hormone using the SRS search program.
How does the SRS search program differ from the NCBI search program that you used today.

Bioinformatics Workshop 2

Sequence Database Workshop

Downloading files from the Internet using your Web browser
Start up your web browser and go to this URL where you can download files using ftp (File Transfer Protocol).

ftp://ftp.ncbi.nih.gov
Find the "gbrel.txt" file and look at it
Do not click on any other file. These are multi-Gigabyte database files and you don't want to download them.
This gbrel.txt file contains the release information for the GenBank database.
Pay attention to the
Size (number of sequences, number of nucleotides, number of species)
Divisions (The database is not a single file, but a collection of files)
In the next part of the workshop, we will be downloading data from sequence databases.
Data Conversion
1. Go to the NCBI Web site
http://www.ncbi.nlm.nih.gov
2. In the Nucleotide database, find the accession number, and download this sequence
Homo sapiens hemoglobin beta chain mRNA complete cds.
There are many hemoglobin sequences in the database. You need to find the specific one that has this description line.
Examine the sequence. Anything look strange for a mRNA sequence?
3. Convert sequences to FASTA format. Why do we need to do this?
4. Translate the RNA into Protein
translate
At what nucleotide should you start the translation?

5. Convert the protein back to RNA (reverse translation or back translation)
backtranslate
What Codon Preference Table should you use? Why do you even need a Codon Preference Table?
Did you get the same nucleotide sequence you started with?
We have software that can answer this question.
Using LALIGN, compare these two nucleotide sequences. We will discuss this program more in an upcoming lecture.

Bio-informatics Workshop 1

These series of workshops are those given to us in our bioinformatics class by Prof Lee Kozar, who is also the director of CMGM at Stanford!

Downloading files from the Internet using your Web browser
Start up your web browser and go to this URL

ftp://ftp.ncbi.nih.gov

Find the "gbrel.txt" file and look at it
This file contains the release information for the GenBank database.
Pay attention to the
Size (number of sequences, number of nucleotides, number of species)
Divisions (The database is not a single file, but a collection of files)
In the next class, we will analyze it more fully and learn how to download specific sequences.

Free software to read sequencing data

These links were given to us by Dr. B after completion of our sequencing experiments.

Chromas sequence viewing software:

chromas11-32.exe (118.426 Kb)

Chromas Lite:

chromaslite201.exe (215.945 Kb)

Link to get sequence scanner:

http://www.appliedbiosystems.com/support/software_community/free_ab_software.cfm

An example of a good summary and critical analysis

This was posted by Dr Claudia Stone as a template for us to follow.

Lammich et al. (2004) Expression of the Alzheimer protease BACE1 is suppressed via its 50-untranslated region.

1) Summary:
· Specific questions
o The entire study focused around whether the 5’-untranslated region of BACE1 mRNA was responsible for translational repression of the BACE1 protein, and specifically what characteristics of the 5’UTR are responsible for the observed repression.
Theoretical context
It is assumed that the cause of Alzheimer’s disease is linked to the collection of the amyloid β-peptide (Aβ).
This is created by the actions of two proteases on the membrane amyloid precursor protein.
γ- secretase
β- secretase
Also known as BACE1
Previous studies.
Vassar, 2002
Mice with targeted deletion of BACE1 do not produce any Aβ.
These mice show no any overt phenotype making BACE1 an ideal drug target.
Fukumoto et al, 2002; Holsinger et al, 2002; Yang et al, 2003
BACE1 protein levels are significantly upregulated in the brains of AD patients compared with non-AD controls.
Yasojima et al, 2001; Holsinger et al, 2002; Preece et al, 2003
These increased BACE1 levels corresponded to unchanged mRNA levels.
Suggests that post-transcriptional mechanisms are at play.
Why pinpoint the 5’UTR as the key?
The structure of this segment is:
446 nucleotides long
GC content of 77%
Three uORFs
All of these characteristics are assumed to be important for the inhibition of translation.
Importance of these questions
Determining mechanisms for the regulation of the BACE1 protein, especially at the translational level, would give ideal targets for future therapy in the progression and prevention of Alzheimer’s disease.
Key experiments with results
Determined whether the 5’ UTR may affect the expression of BACE1.
Expression vectors encoding the ORF of BACE1 alone, with the 5’UTR, or with the 3’UTR were transiently transfected into human embryonic kidney HEK293 cells.
Detection done via immunoblotting of the cell lysate.
Showed the presence of the 5’UTR greatly reduced BACE1 protein levels.
Determined whether the BAE1 5’ UTR could inhibit the expression of a similar downstream open reading frame different from BACE1.
Vectors encoding luciferase with or without the 5’UTR, or an empty control vector, were expressed in HEK293 cells.
Luciferase activity was measured in cell lysates.
Luciferase activity was greatly reduced in cells containing the 5’UTR of BACE1.
Proved that the 5’UTR lowered BACE1 protein levels by selectively reducing the translation of BACE1.
Vectors encoding BACE1, with or without the 5’UTR or empty control vector, were expressed in HEK293 cells.
BACE1 protein was measured by immunoblotting cell lysates.
mRNA levels were measured via northern blotting.
The presence of the 5’UTR had no significant effect on mRNA levels, while simultaneously showing lowered BACE1 protein levels.
Additionally demonstrated that the 5’UTR represses the expression of BACE1 at the translational level.
In vitro-transcribed BACE1 mRNA, with and without the 5’UTR, were translated in a nuclease-treated rabbit reticulocyte lysate.
BACE1 protein was detected without the 5’UTR, however was not observed when using the 5’UTR.
Determined whether the high GC content of the long 5’UTR is sufficient for repressing BACE1 expression or whether the uORFs and their encoded short peptides are required
Mutated the start codon of three uORFs from ATG to ATA
Mutated BACE1 plasmids were transfected into HEK293-APP cells.
BACE1 protein levels were measured in the cell lysate by immunoblotting.
Combined mutations of upstream ATGs showed a slight but significant increase in BACE1 expression compared with single mutations
Reveals that the uORFs account for only partial repression of BACE1 expression
Investigated the effect of several deletion mutants of the 5’UTR of BACE 1 with lowered GC content.
Mutations occurred either at nucleotides 1-223, 224-446, or 1-390 and the expression of BACE1 protein was measured.
Showed that both the 5’- and the 3’half of the UTR have a strong inhibitory effect, with it being more pronounced at the 5’ end.

2) Critical Analysis

The article is a systematic progression of the author’s ideas and logic in the development of the study. The reader is given a clear background discussion to serve as the introduction to the topic and why the author has chosen to formulate such a study. Each experiment is explained in the results and discussion section, and reasoning is given unto why the next experiment should be carried out. Additionally, each experiment is explained concisely, with the necessary specifics laid out in the methods section, allowing the reader to follow the thought process of the author and constantly anticipate the next direction of the study.

There is more than enough evidence supporting the author’s claim that the 5’UTR is responsible for repressing the translation of BACE1 without the need to repress transcription. Most of the experiments were designed to test this hypothesis explicitly, and even when it had been proven with an experiment, the study goes one step further by confirming it with an additional experiment.

The study loses the flow of direct evidence during the discussion that the “GC-rich region of the 5’UTR forms a constitutive transition barrier, which may prevent the ribosome from efficiently translating the BACE1 mRNA.” The author states that the 5’UTR repression is functioning because of either the high GC content, or because of the uORFs. An experiment is conducted that refutes the idea of the uORFs, however the author immediately states that it must because of the GC content creating a tightly folded secondary structure. Although computer modeling (MFOLD program) of the 5’UTR shows that it’s free energy is sufficient for inhibiting translation, no subsequent testing of this ribosome blocking theory is carried out. An additional experiment is carried out which shows that substituting certain regions of the GC-rich sections of the 5’UTR does indeed increase expression of the BACE1 protein. My belief is that the author is using the previous studies of Wood et al, 1996 and Clemens & Bommer, 1999 as support for the ribosome assumption, but without direct reference.

I agree with the author that these studies are important. The mere fact that there are over 24 million cases of dementia worldwide with around 60% due to AD, shows that identifying a specific mechanism and target for therapy could benefit many individuals. Because the specific, and probably varied, cause of the disease remains undiscovered, the ability to block a mechanism this far downstream would negate may factors that reside earlier, such as at the chromosomal level. Thus a treatment designed at this point could be applied to many patients regardless of disease origin.

Summary and Critical Analysis - Sample 1

As part of my Molecular and Cell Biology course work, I have to routinely write summaries and critical analyses of scientific articles. Though not perfect, these few samples may give you an idea of what to put down.

Paper 1: http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=1299076

Summary and Critical Analysis: Expression of the Alzheimer protease BACE1 is suppressed via its 5’- untranslated region

Summary:
BACE1 or Beta site amyloid precursor protein (APP) cleaving enzyme 1 is a membrane protein involved with the secretory pathway, and hence is localized to endosomes, trans golgi apparatus and the plasma membrane. BACE1 is expressed primarily in the brain and the pancreas. Previous research has shown an interesting link between BACE1 and Alzheimer’s disease. It was observed that Alzheimer’s disease patients expressed elevated levels of BACE1 protease. Alzheimer’s disease is presumed to be caused by the formation and aggregation of amyloid β peptides (Aβ). Studies have shown that Aβ peptides are formed by the cleavage of APP by BACE1. It was also noted that despite elevated protein levels, mRNA levels of BACE1 were not affected in AD patients. In attempt to analyze the cause for an elevation in BACE1 levels among Alzheimer’s disease patients, Lammich et al have proposed that the 5’ untranslated region plays a regulatory role in normal adults. Once conclusively proven that 5’ UTR is the defective portion in Alzheimer’s disease, they contend that a cure for the disease can be attempted.
In order to study the role of the 5’UTR in BACE1 expression, the authors first characterized the region and conformed that it is 446bp long, has 77% GC content and contains 3 upstream ORFs. They also presume that the 5’UTR plays an important role as a major part is highly conserved in humans, mice and rats. In their initial experiments, the authors transfected HEK293 cells with BACE1 ORF, BACE1 + 5’UTR and BACE1 + 3’UTR. They observed that BACE1 protein levels were significantly less in cells transfected with BACE1 + 5’UTR. 3’UTR did not seem to play a significant role in BACE1 inhibition. In order to determine the regulatory role of the 5’UTR region, they transfected HEK293 cells with the lucferase ORF and luciferase containing the 5’UTR of BACE1. They observed that cells containing the 5’UTR expressed very low levels of luciferase indicating that the 5’UTR indeed played a role in repressing the expression of a downstream ORF.
In the next portion of their study, Lammich et al proposed that the BACE1 5’UTR affected translation and not transcription. In order to prove this, they first studied the levels of BACE1 proteins in transfected HEK293 cells by immunoblotting and also studied the levels of mRNA by northern blotting. They observed that levels of mRNA were not affected even though BACE1 proteins levels in those cells was reduced up to 40 fold. To further confirm their finding, they repeated the experiment in nuclease treated rabbit reticulocyte lysate and obtained similar results.
In order to show that the results obtained were not cell-type dependant, the experiments were repeated in African green monkey COS7 cells and also in human neuroglioma H4 cells. Similar results were obtained in all the cell types confirming that the regulatory effect of the 5’UTR was cell-type independent. In order to prove that the results were not promoter dependent, the experiments were repeated with two other promoters CMV and EF1α and similar results were obtained.
In order to determine the exact portion of the 5’UTR playing a repressive role, they conducted various experiments with different portions of the 5’UTR deleted. They observed that though deletions in the three uORFs showed increased BACE1 levels, the increase were not as significant as when the 5’UTR was completely absent. Hence, Lammich et al propose that the stable secondary structure of the BACE1 5’UTR rich in GC forms many stem loops which inhibit translation in vitro.
Critical Analysis:
CDC estimates that Alzheimer’s disease is a seriously debilitating illness affecting about 4 million Americans. Being a leading cause of dementia in older adults, there is an urgent need to understand and find a cure to Alzheimer’s. In attempting to solve this problem, Lammich et al have indeed chosen a very important issue.
The paper is very concise and to the point, with ample illustrations of results obtained. Though very clear and concise in most parts, I would have appreciated further explanation about Fig 1B, which I found very difficult to analyze.
The author’s use of COS7 cells and H4 cells in addition to HEK293 cells helps reduce the probability of cell-type influence on the results, but I am unsure if they are sufficient to eliminate all possibility of cell-type effects. More experiments are probably required, using other cell types, like pancreatic cells, to confirm the results obtained. Further studies to analyze the actual structure of the 5’UTR and the mechanism of repression of translation would also prove to be very beneficial.
Another key question not answered in this paper is the role of BACE1 in normal brain cells. The authors mention that BACE1 levels are primarily elevated in the brain and in pancreas, and they also mention a possible secretory role for BACE1, which does not explain the higher occurrence of BACE1 in healthy brain cells.
In conclusion, I believe that this paper is very informative but further research is needed to get closer to understanding BACE1 and the 5’UTR of BACE1 and their role in the prevention and cure of Alzheimer’s disease.

Loading an Agarose Gel, Running it and Analyzing it

Material Required:

Pipettes

Pipette tips

Parafilm

Molecular marker

Loading dye

RNA/ DNA Sample

Ideal loading volumes: 3 to 5μl of sample + 3 to 5μl of loading dye

1 to 2μl of molecular marker + 2 to 3μl of dye

Technique:

1. Place a 1 inch piece of parafilm on the work table.
   
2. Add loading dye.
   
3. To the loading dye on the parafilm, add the DNA sample. Mix well by pipetting.
   
4. Load into a well carefully.
   
5. To load the molecular marker, repeat steps 1 to 4 and replace DNA sample with the molecular marker.
   

Running an Agarose Gel:

Run at 110 to 120 V for 45 minutes to 1 hour. Time will vary. Please look at the position of the leading dye and turn off unit before it runs over.

Imp: While placing the gel, make sure that the wells are on the same side as the black or negatively charged electrodes. Since nucleic acids are negatively charged, they run towards the red or the positively charged electrodes. If the well is placed on the same side as the positively charged electrodes, your sample will run over into the buffer.

Staining an Agarose Gel:

Stain in Ethidium bromide solution for 2 to 4 minutes. Make sure that the stain is on a shaker for efficient staining.

[Since ethidium bromide is a carcinogen, take care while handling it.]

Destain in distilled water or DI water for 15 to 20 minutes.

Nucleic acids will be visible under UV.

Pouring Agarose Gels

Today we used Agarose gels to verify that our DNA isolation was successful. What I learnt about agarose gels:

1. They are very effective in separating large segments of DNA and RNA.
2. They do not have high resolution, i.e., they cannot separate segments which are of very similar sizes.
3. Range of separation: 200 bases to 10 Kilo bases
4. To separate nucleic acids larger than 10 Kb, you need to run them in Pulsed field gels.
5. Always make the agarose in the same buffer as that in which the gel is run.
6. Use of concentrated buffer while making the gel may result in the gel melting during electrophoresis.
7. garose gels are made in a w/v manner - weight of agar in volume of buffer; For example: in order to make 1% Agarose gel, add one gram of agarose to 100ml of buffer.
8. Buffer used by us: 1X TAE (Tris Acetate EDTA)

Protocol: 1% Agarose Gel (Adjust according to percent and volume required)

1. Take a 200 ml beaker.
2. Add 1 gram of agar to 100 ml of 1X TAE, swirl to mix.
3. Microwave, mixing intermittently, till all the agarose is completely dissolved. The solution will appear clear.
4. Cool the solution till the beaker is comfortable to touch. If the agarose is too warm, it might warp the plates.
5. Prepare the plates before heating the agarose solution. Place the comb. Make sure the plates are on a level surface.
6. Pour the agarose solution into the plates.
7. Let sit at room temperature for 30 minutes. If you need the gel quickly, place in the refrigerator for 5 minutes. The gel is then ready to use.
8. Remove the comb prior to use.

Important Websites to know in Bioinformatics

As I was feeling my way through a bioinformatics workshop today, I realized how long it would take me to find these sites by myself with no help from Professor Lee Kozar. So, I decided to quickly jot them down someplace safe so if and when I need them, they are readily available!

Now, the first thing to know in binf (my code for bio-informatics in this blog) is where to search for what

Parameter	Database
Nucleotide	Genbank, EMBL
Protein	SwissProt, PIR, GenPept
3D Structures	RCSB (PDB)
Enzymes	LIGAND
Sequence Motifs	Prosite, Blocks, eMotifs
Pathways and Complexes	KEGG, EcoCyc
Molecular Disease	OMIM
Biomedical Research	Medline
Vectors	UniVec
Protein Mutations	PMD (Protein Mutant Database)
Gene Expressions	GEO (Gene Expression Omnibus)
Microarray Data	SMD ( Stanford Microarray Database)
Chemical Data	MDL

An important ftp site to know to get more information about Genbank

ftp://ftp.ncbi.nih.gov

Important sites for Data Conversion:

Site that translates RNA/ DNA to protein:

http://www.ebi.ac.uk/emboss/transeq/

A site that coverts the protein sequence back to RNA/DNA (Reverse translation or back translation)

http://bioinformatics.org/sms2/rev_trans.html

A site to compare two nucleotide sequences

http://www.ch.embnet.org/software/LALIGN_form.html

Extraction of DNA from a sample

Last Thursday, we had our first lab assignment, which was to extract our own DNA from a salivary sample and use the extracted DNA as PCR template. The following is the bench protocol followed by us. All the reagents used were from Qiagen, and the basic protocol was adapted from Qiagen's DNeasy Blood and Tissue Handbook.

Preparation:

1. Ensure that ethanol has been added to both the washing buffers (AW1 and AW2).
   
2. Set up a water bath at 56° Celsius.
   
3. Check to make sure that there is no precipitation in the Lysing buffer (AL). If there is any, re-dissolve it.
   
4. All the subsequent steps are preferably carried out at temperatures between 15° and 25° Celsius.
   

Procedure:

1. Trying to be as dignified as possible, collect some saliva in a 1.5 or 2 ml microcentrifuge tube. (Ideally, sample should come to about the 0.5 mark)
   
2. Centrifuge at 300 rpm for 5 to 10 minutes or till a firm pellet is formed at the bottom of the microcentrifuge tube.
   
3. Carefully pipet out the supernatant. This can be a tricky process as the pellet is easily dislodged. If the pellet is dislodged, re-centrifuge for 3 minutes.
   
4. Re-suspend pellet in 200μl PBS (Phosphate buffered Saline)
   
5. Add 20μl of proteinase K.
   
6. Add 200μl of buffer AL (Lysing buffer). Make sure that you are not using ATL or tissue lysing buffer at this stage. ATL is much stronger than AL.
   
7. Mix by vortexing, and incubate at 56° Celsius for 10 minutes.
   
8. Add 200μl ethanol (96% to 100%). Mix by vortexing.
   
9. Transfer the mixture into a DNeasy MiniSpin Column in a 2ml collection tube.
   
10. Centrifuge at 8000 rpm for 1 minute.
    
11. Discard liquid collected in the collection tube. If you have lots of collection tubes, you can discard the collection tubes also.
    
12. Place spin column in the emptied or a new 2 ml collection tube.
    
13. Add 500μl of buffer AW1 (Washing buffer 1).
    
14. Centrifuge at 8000 rpm for 1 minute.
    
15. Discard liquid in the collection tube. Changing collection tubes at this stage is once again optional.
    
16. Place spin column in the emptied or a new 2 ml collection tube.
    
17. Add 500μl of buffer AW2 (Washing buffer 2).
    
18. Centrifuge at 14,000 rpm for 3 minutes.
    
19. Remove the spin column carefully so it does not touch the flow through liquid or the collection tube.
    
20. You can discard the collection tube and the flow through at this stage.
    
21. Place the spin column in a new 1.5 or 2 ml microcentrifuge tube.
    
22. Add 200μl of the AE buffer (Elution buffer) to the spin column.
    
23. Incubate at room temperature for one minute.
    
24. Centrifuge at 6000 rpm for 1 minute.
    
25. If you want greater yield, repeat steps 21 to 24 again and combine the eluents from both the microcentrifuge tubes.
    

Conclusion:

• The DNA collected finally in the microcentrifuge can be used as the template for PCR experiments. Add 1μl of obtained eluent to 20μl of master mix and place in a thermocycler.
  

Elutrap electro-elution system

This is another protocol that I wrote for Dr Kim's Lab, which proved to be very useful. When I first started doing some research about the equipment, I found that Schleicher-schuell (the company which initially came up with the elutrap) was acquired by Whatman, and the protocol was no longer published online. I have a saved copy of the old protocol, and if anyone needs it, feel free to get in touch with me.

Protocol: Elutrap Electro-Elution System

Introduction

Elutrap electro-elution system is a membrane trap elution system used for the extraction, concentration and dialysis of DNA and other charged molecules above the size of 5 kDa¹. In our lab, we commonly use the elutrap to extract DNA and RNA from polyacrylamide gels. The advantages of this system over previously used techniques such as diffusion and simple electro-elution are a) high recovery rate, b) high reproducibility, c) high purity of recovered material and d) the ease of use¹. Elutrap works by forming a collection chamber bordered by two membranes, a BT2 membrane and a dialysis membrane, into which buffer ions and target molecules less than 3 – 5 kDa collect under the influence of an electric field.

Materials & Methods

Equipment:

Elutrap device: consisting of sample chamber of 20ml capacity

Electrophoresis chamber with a central 4-channel tray insert

BT2 membrane – one per each device used

BT1 membrane – one per each device used

Dialysis membrane – one per each device used

Open ‘U’ inserts – 3 per each device used

Closed ‘U’ inserts – 2 per each device used

0.5X TBS (Tris Borate EDTA) Buffer – 2 liters

Ultrapure TBE buffer – about 15 ml per device used

Tweezers

Scissors

Gel pieces containing the purified template

A small beaker containing DI water – to place dialysis membrane prior to use

Kim wipes

Toggle lever to tighten the screws

Assembly and Running of the Elutrap System:

1. Clean work area and place kim wipes.
2. Wash all equipment with hot, distilled and then with DI water.
3. Cut out an adequate piece of dialysis membrane and place in the beaker containing the DI water. Remember that the dialysis membrane is bi-layered. We however use a single layer in the device. Hence, two filters can be prepared from one piece of the dialysis membrane. Remember to cut the dialysis membrane with one edge slightly higher than the other similar to the shape of the BT1 membrane.
4. Loosen the screws of the elutrap device. Place the U shaped inserts into the two chambers of the device. The larger chamber should hold 4 inserts – three open and one closed. The smaller chamber should contain a single closed insert. Make sure that the inserts are tightly placed and rest on the designated slot. The notched surface of the inserts always faces the central elution chamber containing the gel pieces. Tighten the screws lightly.
5. Place the dialysis membrane between the screw and the closed U insert in the large chamber of the device. It is very important to place the sloping side of the dialysis membrane next to the triangular mark on the side of the elutrap device.
6. Place the BT2 paper filter on the other surface of the closed U insert while taking care to keep it dry. Tighten the screw. To check for the proper placement of the two membranes, place the device upright with the dialysis membrane on top, add some DI water and check for signs of leakage. Leakage can be detected by the appearance of water on the paper filter. Keep the dialysis membrane moist till the placement of the BT1 membrane. The BT2 paper filter prevents movement of gel pieces and large impurities into the collection chamber.
7. The size of the collection chamber formed by the dialysis membrane and the BT2 membrane can be varied by altering the position of the placement of the latter membrane. If extracting large amounts of DNA or RNA, place the BT2 membrane between two open U inserts.
8. Take the BT1 membrane out using forceps and clean thoroughly with DI water to remove all traces of glycerol. Place it between the screw and the closed U insert in the smaller chamber of the elutrap device. It is very important to place the sloping surface of the BT1 membrane next to the triangular mark on the side of the device.
9. Fill the electrophoresis chamber to half full with 0.5X TBE. Place the elutrap device into one of the channels of the tray insert of the electrophoresis chamber. The collection chamber end of the device should be placed near the open aperture at the end of the channel towards the positively charged electrode.
10. Place the tray against the tray stop so that the bottom aperture on the tray lies over the buffer. Align the tray such that the two apertures lie on top of each other allowing the passage of current into the channel. When the channels are not occupied with a device, make sure the apertures do not align, turning off the flow of current through them.
11. Carefully add 0.5X TBE buffer to the electrophoresis chamber while simultaneously adding 0.5X ultrapure TBE to the central elution chamber of the elutrap device.
12. Add the gel pieces to the central elution chamber containing the 0.5X ultrapure TBE. Take care not to clump the gel pieces together. Do not crowd the gel pieces on either end of the elution chamber. Also make sure that the gel pieces are completely covered with the buffer.
13. The buffer in the electrophoresis chamber should cover the electrodes. The buffer in the elution chamber should be about one mm from the top.
14. Plug in the electrodes. Remember that the red colored positively charged electrode is on the same end as the collection chamber.
15. Run the unit at 150V for three hours. Collect the buffer containing DNA/RNA every hour from the collection chamber. Then, run the unit at 50V overnight for the final collection. Each collection should typically yield between 200 to 800μl of the buffer.
16. To collect, hit the “run” switch to pause the electrophoresis. Switch the electrodes so that the negatively charged black electrode faces the collection chamber and run for 20 seconds. This separates DNA/RNA from the dialysis membrane. Switch the cables again immediately so you don’t run the unit on reverse.
17. Set a pipette to 200μl. Collect using a loading tip, moving the tip carefully along the edges to recover the entire sample. Take care not to damage the membranes during collection. In case of inadvertent piercing of the membrane, save the buffer. Sample can be extracted from the buffer by ethanol precipitation and lyophilization.
18. Collecting the sample too slowly can result in collection of a greater volume of the buffer as the buffer continues to diffuse into the collection chamber. Careful quick collection is desirable.
19. After collection of each sample, check the OD (optical density) of the buffer collected. The OD of subsequent collections should be lower as the concentration of the sample gets diluted with the buffer. Most of the nucleic acid is typically obtained from the first collection.
20. Remember to check that the electrodes are appropriately placed and to hit the “run” switch after each collection. Turn off the unit after completion of the elution.
21. Following completion of the electro-elution, proceed with ethanol precipitation.

Conclusion

1. Organic solvents may damage the device and should be removed promptly.
2. The membranes should only be handled with gloves on to avoid RNase contamination. The same applies to handling of the equipment. If you suspect contamination of the equipment with RNase, wash with 0.05M NaOH, followed by 0.05M acetic acid. Finally, rinse thoroughly with DI water.
3. To make the buffer RNase free, add DEPC (diethylpyrocarbonate) to 0.1% and allow the solution to stand for 12 hours at 37°C. It is essential to then autoclave the buffer to remove all traces of DEPC.
4. Always keep the BT1 and the dialysis membrane moist. However, take precautions to keep the BT2 membrane dry for as long as possible.
5. Gel slices should not extend beyond 6 cm in the sample chamber. Do not stack gel slices above the device height. Take care not to crush the gel as this may impair elution efficiency.
6. To clean, discard the buffer down the drain. Discard all membranes. Wash well with warm water, followed by distilled water and DI water.

References:

1. Gobel, U “Quantitative electroelution of oligonucleotides and large DNA fragments from gels and purification by electrodialysis” Journal of Biochemical and Biophysical Methods 1987 August ;14(5):245-60.
2. Schleicher & Scheull - Protocol: Elutrap electro-elution system

Pouring Polyacrylamide Gels

Last Summer, I worked in the research lab of Dr. Chul Kim where I wrote this protocol for his lab.

Preparation & Usage of Polyacrylamide Gels

Introduction

Polyacrylamide gels are used for the separation of proteins or small nucleic acids. Larger nucleic acids are typically separated using agarose gels. There are two types of polyacrylamide gels used in the laboratory. One type is the denaturing polyacrylamide gels which have urea added. Urea acts as the denaturant and ensures separation of the strands during migration. Typically 20% polyacrylamide gels are used as denaturing gels in our lab. The other types of polyacrylamide gels used are Native gels. These gels do not contain urea and are used to observe the separation of molecules due to conformational differences. Typically 12% polyacrylamide gels are used as native gels in our lab. Since oxygen inhibits the polymerization process, these gels are poured between glass plates.

Materials & Methods

Equipment required:

Measuring cylinder for measuring acrylamide

Micropipettes and micropipette tips

Hot plate with stirrer

Stir bar

Beaker

2 Glass plates (one slightly smaller than the other) and spacers

Gel sealing tape (for small and long gels)

Comb

Binder clips

Casting clamp (for big gels)

Power Supply

Material required (all must be kept refrigerated at 4^oC):

Acrylamide: 12% (without urea) or 20% (containing urea)

10% Ammonium persulfate (APS): catalyst in the polymerization of acrylamide

N,N,N',N'-Tetramethylethylenediamine (TEMED): adjunct catalyst for the polymerization of acrylamide

Quantities required:

Small Gel (~ 20 cm x 20 cm): Acrylamide: 50 ml

APS: 666μl

TEMED: 50μl

Long Gel (~ 35 cm x 20 cm): Acrylamide: 125ml

APS: 1665μl

TEMED: 125μl

Big Gel (~ 41 cm x 33 cm): Acrylamide: 220ml

APS: 2930μl

TEMED: 220μl

Standard Protocol: Acrylamide: 75ml

APS: 1ml

TEMED: 75μl

Method:

1. Clean equipment with hot, distilled and de-ionized water and assemble.
2. Prepare the glass plates:

Place spacers on the longer plate and place the smaller plate on top. If smaller plate has a bevel, point bevel facing inwards on the comb bearing side of the plate. [Make sure that the spacers and the comb are all of the same thickness, 1.6mm.]

Ensure that the spacers and plates are properly aligned and square, the foam pads are pressed tightly to the lower plate and clip the sides with binder clips for small and long gels. Tape the lower end carefully to prevent leakage while pouring for long and big gels, and place a spacer to close the lower border of the small gel. For big gels, close sides using casting clamps.

3. Crumple up paper towels and place beneath corners of plates to catch polyacrylamide leakage.
4. Place an appropriately sized beaker containing a stir bar on a hot plate and set the stir to 8 (high intensity stirring).
5. Take acrylamide from the refrigerator, shake well to mix, and add required quantity to the beaker. Place the acrylamide back before taking the next reagent. [Important: Use the yellow acrylamide measuring cylinder only for the acrylamide]
6. Take APS from the refrigerator, shake well to mix, and add required quantity to the beaker. Replace the APS before taking the next reagent.
7. Take TEMED from the refrigerator, shake well to mix, and add required quantity to the beaker. Replace.
8. Working as quickly as possible, incline the glass plates to a sharp angle and pour the acrylamide mixture continuously into the glass plates along the edge from corner to corner. Take care not to form bubbles while pouring.
9. Once glass plates are filled, place comb while taking care not to push the comb in completely leaving about a mm (pushing the comb in completely causes a suction resulting in the breakage of the gel during removal of the comb). Also make sure that the comb is placed in the center.
10. Place the plates horizontally and let the gel solidify. This typically takes around 20 minutes, minimum (Mol. Clon. Advises one hour). Use the remaining polyacrylamide in the beaker as your guide; if it is solid then your gel is solid. Label plates.
11. These plates can be used immediately or stored at room temperature for 24 hours or at 4°C for 48 hours. To store, do not remove tape or comb, surround top of the gel with paper towels dampened with 0.5X TBE and wrap entire gel with plastic wrap.

Running a Small Gel:

Material Required:

0.5X TBE: 1000 ml

Small electrophoresis chamber, cover and cables

Two binder clips

Electrical unit to set parameters for running the gel

Vaseline

Method:

1. Clean the electrophoresis chamber with hot, distilled and de-ionized water (DI-water).
2. Make up 1 liter of 0.5X TBE by adding 100 ml of 5X TBE and 900 ml of DI-water. Cover with parafilm. Label.
3. Take the poured gel; remove the lower spacer and the comb. Trim any edge, if needed. Wash the wells with DI-water to remove particulate matter. Leave the side spacers in place.
4. With the smaller plate facing inwards, place the plates so they rest snugly against the cushioning. Hold them in place using one binder clip on each side making sure the clips seal the lower edge.
5. Mix the prepared 0.5 X TBE solution. Pour it into the upper chamber to top the gel by about a centimeter. Wait at least 5 minutes to ensure that there are no leakages. If there are any leakages, re-clip the plates and try again. You might also want to change the clips and use tighter binder clips.
6. Once you make sure that there is no leakage, incline the unit and pour 0.5X TBE into the lower chamber. This prevents the formation of air bubbles along the gap created by the removal of the lower spacer. Make sure the lower end of the gel is completely immersed and there are no bubbles below the plate. Bubbles formed on the lower edge can result in improper conductance of charge across the gel.
7. Once the lower chamber is ready, top the upper chamber off so the level is at least two cm above the gel.
8. Map out how you are going to load the wells in advance. When possible, take care not to create a pattern that would look the same. This will allow for the identification of samples even if the gel becomes inverted during the staining process.
9. Add an equal amount of loading buffer with dye to your sample (make sure you pick the right one – native or denaturing, and not the concentrated dye). Heat your sample at 90°C for three minutes.
10. Working as efficiently as possible, rinse out the wells with 0.5X TBE using a syringe. Take care NOT to poke the gel with the needle. This removes any traces of urea from the wells. Leaving urea in the wells will result in improper migration of the sample. As soon as the wells are washed, load 20μl of your sample containing the loading buffer dye into each well. Unless you have very good control over the pipette, push the sample only to the first depression of the pipette. Do not push to the second depression point as you may create a bubble that could flush your sample out of the well.
11. Close the electrophoresis chamber. Label. Plug in the red positive cord to the lower electrode and the black negative cord to the upper electrode. As both RNA and DNA are negatively charged, we run them towards the positive charge.
12. Run the gel at a constant voltage of 300V. Let the gel run for around three hours, until the leading dye is within a cm of the lower border of the gel. Turn off the power and disconnect apparatus.
13. Carefully drain the buffer into the lower chamber and remove the gel plates. Remove the spacers. Separate one glass plate from the gel.
14. Fill a container reserved for staining gels to 1/4^th with toluidine blue stain. Carefully ease the gel into this container.
15. Place the container on a shaker set at low speed. Gentle movement of the stain will allow for better incorporation of the dye into the gel. Let the gel stain for a minimum of one hour or until you can clearly make out that the sample has taken up adequate stain. If staining is not adequate, add a few drops of concentrated stain to the container and let stain longer.
16. Pour the toluidine blue stain carefully into a beaker and pour it back into its original container. Toluidine blue is reused so make sure that it does not get contaminated with polyacrylamide gel pieces. Make sure that you don’t dilute the stain. Hold the gel stable with one hand while pouring the stain to prevent the gel slipping out of the container.
17. Cover the gel with DI-water and place back on the shaker. Keep replacing the water periodically to de-stain efficiently. Failure to change the water may result in the stain settling back on the gel.
18. Once the gel is de-stained, proceed with scanning.

Running a Long Gel:

Materials Required:

Large electrophoresis chamber and cables

Electrical unit to set parameters for running the gel

1 liter 0.5X TBE

Vaseline

Method:

1. Clean the electrophoresis chamber with hot, distilled and de-ionized water (DI-water).
2. Make up 1 liter of 0.5X TBE by adding 100 ml of 5X TBE and 900 ml of DI-water. Cover with parafilm. Label.
3. Take the poured gel; remove the tape and the comb. Trim any edge, if needed. Wash the wells with DI-water to remove particulate matter.
4. With the smaller plate facing inwards, place the plates so they rest snugly on the cushioning. Turn the knobs to hold the plates firmly in place.
5. Make sure the knob connecting the upper and the lower chambers is closed.
6. Mix the prepared 0.5 X TBE solution. Pour it into the upper chamber to top the gel by about two cm. Wait for 5 minutes to ensure that there are no leakages. If there are any leakages, remove the buffer and try to seal the leak with Vaseline.
7. Once you ensure that there are no leakages, pour 0.5X TBE buffer in the lower chamber till the lower end of the gel is completely immersed; about 2 cm above the gel.
8. Map out how you are going to load the wells in advance. When possible, take care not to create a pattern that would look the same. This will allow for the identification of samples even if the gel becomes inverted during the staining process.
9. Add an equal amount of loading buffer to your sample (make sure you pick the right one – native or denaturing, and not the concentrated dye). Heat your sample at 90°C for three minutes.
10. Working as efficiently as possible, rinse out the well with 0.5X TBE using a syringe. This removes any sediments of urea from the wells. Leaving urea in the wells will result in improper migration of the sample. As soon as the wells are washed, load the sample into your well.
11. Make sure that you do not poke the gel with the loading tip. If the tip gets clogged, remove, trim the end using a sharp scissors and continue loading.
12. Plug in the red positive cord to the lower electrode and the black negative cord to the upper electrode. As both RNA and DNA are negatively charged, we run them towards the positive charge. Label the unit.
13. Run the gel at a constant voltage of 300V. Let the gel run for around seven hours, until the leading dye is within a cm of the lower border of the gel. Turn off the power and disconnect apparatus.
14. Open the knob connecting the upper and lower chambers. Carefully drain the buffer into the lower chamber and remove the gel plates. Remove the spacers. Separate one glass plate from the gel.
15. Fill a container reserved for staining gels to 1/4^th with toluidine blue stain. Carefully ease the gel into this container.
16. Place the container on a shaker set at low speed. Gentle movement of the stain will allow for better incorporation of the dye into the gel. Let the gel stain for a minimum of one hour or until you can clearly make out that the sample has taken up adequate stain. If staining is not adequate, add a few drops of concentrated stain to the container and let stain longer.
17. Pour the toluidine blue stain carefully into a beaker and pour it back into its original container. Toluidine blue is reused so make sure that it does not get contaminated with polyacrylamide gel pieces. Make sure that you don’t dilute the stain. Hold the gel stable with one hand while pouring the stain to prevent the gel slipping out of the container.
18. Cover the gel with DI-water and place back on the shaker. Keep replacing the water periodically to de-stain efficiently. Failure to change the water may result in the stain settling back on the gel.
19. Once the gel is de-stained, proceed with scanning.

Running a Big Gel:

Materials required:

Large electrophoresis chamber and cables

Electrical unit to set parameters for running the gel

1 liter 0.5X TBE

Vaseline

Method:

Clean the electrophoresis chamber with hot, distilled and de-ionized water (DI-water).

Make up 1 liter of 0.5X TBE by adding 100 ml of 5X TBE and 900 ml of DI-water. Cover with parafilm. Label.

Take the poured gel; remove the tape and the comb. Trim any edge, if needed. Wash the wells with DI-water to remove particulate matter.

With the smaller plate facing inwards, place the plates so they rest snugly on the cushioning. Turn the knobs to hold the plates firmly in place.

Make sure the knob connecting the upper and the lower chambers is closed.

Mix the prepared 0.5 X TBE solution. Pour it into the upper chamber to top the gel by about two cm. Wait for 5 minutes to ensure that there are no leakages. If there are any leakages, remove the buffer and try to seal the leak with Vaseline.

Once you ensure that there are no leakages, pour 0.5X TBE buffer in the lower chamber till the lower end of the gel is completely immersed; about 2 cm above the gel.

Map out how you are going to load the wells in advance. When possible, take care not to create a pattern that would look the same. This will allow for the identification of samples even if the gel becomes inverted during the staining process.

Add an equal amount of loading buffer to your sample (make sure you pick the right one – native or denaturing, and not the concentrated dye). Heat your sample at 90°C for three minutes.

Working as efficiently as possible, rinse out the well with 0.5X TBE using a syringe. This removes any sediments of urea from the wells. Leaving urea in the wells will result in improper migration of the sample. As soon as the wells are washed, load the sample into your well. The large well can contain a maximum of 2 ml of sample + dye. Use a 300μl gel loading tip and load sample in increments in a layering motion. Make sure that you do not poke the gel with the loading tip. If the tip gets clogged, remove, trim the end using a sharp scissors and continue loading.

Plug in the red positive cord to the lower electrode and the black negative cord to the upper electrode. As both RNA and DNA are negatively charged, we run them towards the positive charge. Label the unit.

Run the gel at a constant power of 45 Watts per each big gel for approximately 6500 Volt hours. With this setting, the gel is usually allowed to run overnight.

Turn off the power and disconnect apparatus.

Open the knob connecting the upper and lower chambers. Carefully drain the buffer into the lower chamber and remove the gel plates. Remove the spacers. Separate one glass plate from the gel. Take a large sheet of plastic wrap and carefully ease the gel onto the wrap.

Place the wrap on reflective squares which fluoresce when irradiated with short wavelength UV rays.

Identify the sample and cut the gel into 0.5cm pieces using a clean scalpel. Transfer the pieces into a clean falcon tube using a clean forceps. Label and store at -20°C until ready to extract the sample using an ELUTRAP.

Conclusion

1. Acrylamide is a neurotoxin. Hence, all work should be conducted wearing gloves. Special care should be taken to ensure that the appropriate measuring cylinder alone is used. Acrylamide [liquid] should never be disposed down the sink.
2. Polyacrylamide or the polymerized acrylamide is not a toxin, and can be discarded in the trash.
3. If unsure about the procedure, polymerization time can be delayed by placing the acrylamide, APS, TEMED mixture on ice.
4. After running the gel, TBE buffer used can be discarded down the sink.
5. Toluidine blue stain is reused. Place back into original container after use. DI-water containing stain can be discarded down the sink.

References

“Molecular Cloning: A Laboratory Manual”, Second Ed., by J Sambrook, E.F. Fritsch and T Maniatis (1989) 12.74 – 12.80

http://www.vivo.colostate.edu/hbooks/genetics/biotech/gels/principles.html

Introduction

Like all good beginnings, I would like to introduce myself and my work. I am a Master's student in Cell and Molecular Biology, and am also currently doing a certificate program in Biotechnology. My undergraduate degree was in Dentistry. After completion of my Dental degree from SRMC & RI, I came to California, where I completed one year of Post-baccalaureate studies in Cell and Molecular Biology. On completion of the above, I was fortunate enough to be accepted into the Master's program at Cal State, East Bay, which brings us up to date.

My Mentor: Every person has an idol, a guru, and mine is Professor Chris Baysdorfer. An untiring perfectionist, his ability to go from class to class and from topic to topic leaves all of us amazed.

In the subsequent posts, I will try to put my experiences, projects and reviews online. Since I am currently taking the following four courses, my posts in the next few months will cover these topics mostly.

Courses lined up:
PCR, DNA seqencing and Fragment analysis
Cell and Molecular Biology
Bioinformatics
Molecular Biology Seminar