BLOOD storage and PCR problems! HELP - (Dec/23/2007 )
It is my task to amplify a 909bp segment from human genome. First, I extracted DNA from whole blood samples, then I amplified them following an article. However I never get anything for nearly two months now!!!
My questions are:
- What is the amount of EDTA as anticoagulant? I used 1 volume of 10mM EDTA (pH adjusted by NaOH) for every 5 volume of whole blood, is it too much???
- Is it necessary to use anticoagulant? Some friends told me that they just keep the samples in -20 for weeks. I suppose it depends on how long the samples are stored.
- How does heparin interfere with the PCR? People keep telling me not to use heparin, but I do not understand why???
- I think that DNA are only in the nucleated white blood cells, so why do people talk about red blood cell lysis? and if we want human DNA (not virus), why do we use serum? I suppose when we centrifuge, WBCs precipitate
- About the PCR, the primers are designed with Tm of 53 and 48.6, but in the article the annealing temperature is 56. I once have a faint band with that 56, but that never happens again, so I tested 49 and 52. Sadly, there were no bands at all!
- One last question, anyone try PCR from boiled blood? I really need some advices now.
Santa Claus, where are you, help me!!! (