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

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.

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.