Hi there. I am experiencing serious problem with PCR analysis .I am not very keen in the field so please give me some hint what might get wrong. I have been using the same protocol for several times since last years and suddenly I started getting no products in the electrophoresis. I changed TAq, buffer, calibrate the thermocycler, used different pair of primers no response. I ordered new dNTPs and finally I got once a band only of the cultured bacteria in PCR water and not any at all bands in my extractions. These extractions used to work very well in previous PCRs. What might be wrong? Please help...I cannot find a solution..I do not belive that all extractions have degradaded...
Thanks

Since you have tried
TAq, buffer, calibrate the thermocycler, used different pair of primers. dNTPs and new DNA extracts

you may want to try
1. change the annealing temp
2. see if the primers are degraded/GC rich/secondary structured
3. try hot start PCR
4. work on a different PCR machine
5. run a gel of your extracts to make sure
6. ask a labmate work on the same PCR to see if he/she gets anything

Hi there. I am experiencing serious problem with PCR analysis .I am not very keen in the field so please give me some hint what might get wrong. I have been using the same protocol for several times since last years and suddenly I started getting no products in the electrophoresis. I changed TAq, buffer, calibrate the thermocycler, (clue 1) used different pair of primers no response. I ordered new dNTPs and finally I got once a band only of the (clue 2)cultured bacteria in PCR water and not any at all bands in my extractions. These extractions used to work very well in previous PCRs. What might be wrong? Please help...I cannot find a solution..I do not belive that all extractions have degradaded...
Thanks.

clue 1: other primer also not work; clue 2: nothing on ur extracted DNA but PCR water with bacteria have band. ALL of this sum up to PCR INHIBITORs!!!!!

can u post what is ur extracted DNA concentration and purity. And how much u volume of extracted DNA you load per PCR reaction.

Ok here is an easy way to test. let us assume that you load say 3ul of extracted DNA. Do you have labmate who have working PCR? If yes then in that reaction replace 3ul of PCR water with ur extracted DNA. If the result suppose to be positive but in gel show negative mean that ur extraction may have high PCR inhibitor.

And of course too much DNA will inhibit too. If you grow bacteria in different broth media maybe you can wash it few times with saline before process for extraction. please keep update. and do tell us whether is inhibitor or not.