I used HotShot protocol to extract DNA from pig ears, needing to do PCR RFLP. Most of my samples I had successful PCR first time, and succesfsul RFLP. For some samples I only get very faint bands, even after extracting the same animal a second time. Most recently I played around with adding 0.5ul for samples with a concentration between 100-200 ng/ul, and add 1.0ul for those with concentration 50-100 ng/ul, to a 25ul reaction volume. The 260/280 is low, around 1.2-1.6. I know my primers, reagants, temperatures etc all work, it seems to just these last 30 samples are finicky. What can I do to get better amplification using this "dirty" DNA?
You probably have pcr inhibitors present, These can often spoil the reaction by removing or chelating Mg ions essential for the enzyme to work. For samples that do not work I would double the amount of Mg and amplify less dna. Run 1/2, 1/4 and 1/8 amount of dna. Often less dna means less inhibitor so more amplification. Add a couple of extra pcr cycles as well will do no harm
If that fails then re purify the poorly performing dna.
You can test for inhibitors by adding some poorly performing dna to a good dna and amplifying. Use the good dna only as a control. If the added dna spoils the reaction then you have pcr inhibitors present in the poor sample
You probably have pcr inhibitors present, These can often spoil the reaction by removing or chelating Mg ions essential for the enzyme to work. For samples that do not work I would double the amount of Mg and amplify less dna. Run 1/2, 1/4 and 1/8 amount of dna. Often less dna means less inhibitor so more amplification. Add a couple of extra pcr cycles as well will do no harm
If that fails then re purify the poorly performing dna.
You can test for inhibitors by adding some poorly performing dna to a good dna and amplifying. Use the good dna only as a control. If the added dna spoils the reaction then you have pcr inhibitors present in the poor sample
Are you working with a commercial PCR master mix or adding MgCl2, PCR buffer and Taq polymerase separetely in your reactions?
Have you tested adding bovine serum albumin (BSA) in the PCR? This works by chelating organic inhibitors that may be present in low-quality samples. Adding extra cycles could also be useful as Dr. Paul mentioned.
Hey, try to incorporate the Dr. Paul's suggestion.
Sometimes adding extra units of taq DNA pol also works.. try that as well!
All the best !
Hi, Rachel.
Dr Rutland and others already gave you nice ideas. The only other thing I can figure out for you is to perform a simple sodium acetate and ethanol precipitation to get rid of the contaminants which may be interfering with your PCR-RFLP.
Give it a try!
All the best.
Hello Rachel,
I fully agree with Dr. Paul Rutland also you can precipitate as Adriano suggested your sample by sodium acetate and ethanol and so get rid of contaminants but keep in mind that the overall conc of the sample will be a bit reduced. Try to employ these sugestions and hopefully will work.
All the best,
Stefania M. Mang
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