Difference between revisions of "PCR your DNA Workshop"

Revision as of 14:54, 14 February 2016

How do we go from showing the existence of DNA (strawberries, soap and alcohol) to the popular imagery of DNA gels?
This is a consolidated practical go-to version of workshops we have done as Hack-a-Taq and Citizen Science @ Bern. It highlights also the generic lab equipment. You can find similar protocols in other places - the OpenPCR site writes why we may want to PCR some DNA. This is our documentation - we put together the "kit" and "home-made" protocols.

Workflow

With a group of 15-20 people with everyone participating, we see that this workflow can easily take the entire day.
You can stop and continue another time - after the DNA extraction and after the PCR.

1. Get Samples
2. Extract the DNA (PAUSE)
3. Amplify the gene of your interest using PCR (PAUSE)
4. Separate the PCR products by agarose gel electrophoresis
5. Visualize the PCR products (your amplified DNA bits)

What you need to Prepare

First, you need to decide what you will be looking for.
In the Bern Citizen Science Workshop, we decided to take samples from our fellow participants (human cheek epithelial cells) and amplify the serotonin transporter genes to look at the different (Long and Short) forms.
For the Hack-a-Taq weekend, we wanted to see if we had any coliform contamination in the waters, and so we used primers to look for the bacteria.
Designing appropriate primers should be another section - but for many things, there are already primers reported in the literature. You can always start with google searches for PCR primers and the name of your gene of interest. For example, the coliform bacteria primers, we decided upon reading many papers that already checked for its efficacy and specificity.

Sample Preparation

Materials

• sterile (or new pack) of Q-tips or tooth picks or mini-tongue depressors - (anything you can scratch, and that you can autoclave)
• sterile eppendorf tubes, or equivalent plastic or glass tubes - if you want to concentrate your sample by centrifuging, you need centrifuge compatible tubes
• PCR grade water - or autoclaved de-mineralized/distilled water

Equipment

• Optional Centrifuge to concentrate sample

DNA Extraction

'Materials For bacteria and animal cells:

• Genotyping "Kit" - contains extraction buffer to disrupt the membranes and Proteinase K to digest any proteins bound to DNA. There are many genotyping kits out there - in Bern, we used one of these commercially available kits
  
• - or Make your OWN DNA Extraction Buffer - from PCR_your_DNA_Workshop#References Goldenberger et al.:
  • 50mM Tris-HCl pH 8.5 (to buffer the pH)
  • 1mM EDTA (to chelate any divalent cations and prevent DNA digesting enzymes to degrade the DNA)
  • 0.5% SDS (surfactant to burst open the cell and in eukaryotes, nucleus walls)
  • 200ug/ml Proteinase K (to digest any proteins bound to the DNA)

Quick and dirty bursting of cells for bacteria:

• Distilled water

Equipment

• PCR machine or equivalent temperature controlled water bath

PCR

Agarose Gel Electrophoresis

Gel Visualization

NOTE: autoclaving - if you don't have access to a lab-grade autoclave, use a pressure cooker and let it cook for 20 minutes after the pressure is up. To be official, it has to reach 120oC, 15psi for 15 min. If you are autoclaving, make sure not to close the bottles all the way (it will explode), and make sure not to fill any bottle with liquids more than 3/4 full (it will overflow).

Step by Step Procedure

Sample Preparation

• To pellet any bacteria in water samples, centrifuge 10 min at 12,000xg

DNA Extraction

PCR

Basics of PCR can be found explained in many places. The wikipedia PCR page has extensive documentation. If you just want to conceptually visualize what we are doing, here is a video from youtube.

Agarose Gel Electrophoresis

Gel Visualization

References

Goldenberger, Perschil, Ritzler and Altwegg, A Simple "Universal" DNA Extraction Procedure Using SDS and Proteinase K Is Compatible with Direct PCR Amplification. Genome Res 1995 pp. 368-70 --- pdf