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