Problem amplifying DNA from tissue - (Jan/22/2006 )
hi there. could anyone give me some guidelines when developing a Pcr master mix for DNA isolated from tissue(paraffin embedded tissue). Im having great difficulty as the primers i have optimised with Blood DNA is highlighting up, whilst that for the DNA from tissue is not. Is this a common problem as its my first time dealing with DNA tissue. Are there any steps to follow. Ive tried increasing MGCL2 and decreasing the temp, although there is not much variabliltiy in decreasin the temp as the temp differnce is between 55 and 57. Whats more frustrating is that ive speced the dna and gotten good readings , but no DNA amplifies up. And when i did run a crude DNA gel run, nothing no bands appeard. What am i to make of this
You are running the positive control with blood DNA simultaneously?
Of course, the genome is not different in blood vs. tissue, so there must be some inhibitor carrying over from paraffin removal step... if you have enough DNA then try doing an additional phenol chloroform extraction and ethanol precipitation before using as template...
Changing the MgCl2 or temp wont matter, your positive control (the blood sample) works which means the PCR conditions are appropriate (should work on any template containing these sequences) this indicates the problem is in the sample, either with the amount of template, the degradation state of the template or, as I would think is most likely, with the purity of the sample, ie: carryover of inhibitors from deparaffin step or other step...
I've done a lot of work with PET samples, and I find that the DNA is often so fragmented that it is best to keep PCR amplicons less than 400-500bp or amplification becomes difficult
I agree with John. Have you tried reamplifying your samples? I did work with paraffin embedded tissues with a 500 bp amplicon and always had to reamplify to see something in my agarose gel. If you try this be VEEERRRRYYYY careful with contamination!!!
Hope it helps.
There are some good replies here but it may be worth spiking the DNA tissue DNA with the blood tissue DNA and seeing whether you can get a band. This should show whether the PCR is capable of working in the DNA tissue sample.
I also worked in lab once where they were trying to perform Q-PCR on paraffin embedded samples and there was the concern raised the samples would be too degraded. I think it would be a good idea to keep the amplicon as small as possible