Hello, I am working with plant bacteria from capsicum,mostly Pseudomonas. I tried doing PCR with primers of Yamamoto (1995), but it didn't works. I am using now another primers from Agaras and Valverde (2018), but it didnt't works. We use safegreen master mix, and ADN Diluited (20-30ng/ul), I don't know which is the problem? anybody help me? I'm looking suggestions with my PCR. Thanks a lot
Do you have a good positive dna control for your PCR to check that the assay is working
The bacterial dna is probably only a minor amount of the dna. Are you using nested pcr. If not how many pcr cycles are you running and is this end point pcr ?
It is possible that your plant dna contains pcr inhibitors ( measure the OD260/280 and 260/230) and try running less DNA ( so less inhibitor) and a couple more cycles of pcr. Try running dna at 1/2, 1/4 and 1/8th of the amount you are using now
Hello Angela Rivera, firstly what I can tell you from my experience is never expect primer sequences from the literature to work! No disrespect to the fellow researchers but it is always best to blast the sequences again and confirm if the mentioned sequences correspond to the specified gene only. Another important requirement is to set up an annealing temperature using gradient PCR. Although the literature might state a PCR program to be used with the sequences it is best to set up a gradient PCR to check what works for you, since the reagents and instruments vary from lab to lab.
Doing the following might help you solve your problem.
1. Blast the primer sequences and confirm their specificity.
2. Run a gradient PCR to set up the annealing temperature.
3. Importantly, as Paul Rutland mentioned use a known positive control in the assay. Also include an internal control (such as 16S rDNA) to make sure that amount of DNA you are using is enough as well as to check the integrity of the sample.
Hope this helps.
Best!
Dear Paul Rutland
I am working with conventional PCR, and dna from bacteria cultures, I think that dna is in low quantity. Thank you
Dear Angela Rivera . With small amounts of dna you cannot run huge numbers of cycles because misprimed bands will also amplify so I would make a new primer fully inside one of your primers and run 30 cycles of outer f and outer r then take 1ul of the crude pcr product,dilute it 1:100 and re amplify 1ul of diluted dna with inner f and outer R for 25 cycles. Even better is fully nested with inner f and inner R but hemi nested usually works well. If you get a smear on the agarose then run fewer second round cycles
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