Hello,
I am studying DNA barcode (COI) on amphipods but so far, I can´t achieve product PCR+. I have used three primer (Folmer/HCO2198 LCO1490 – Zplank/ZplankF1 ZplankR1- JG/jgHCO2198 jgLCO1490), parameter different of annealing temperature, +/- DNA concentration, + Mg+2, Trehalosa (potent PCR enhancer) to 5%. I used two extraction method DNA: 1) Hot-shot and 2) Wizard SV Genomic DNA. The amphipods are marine (Stenothoidae, podoceridae, maeridae and caprellidae). The concentration of DNA is between 5 ug/uL to 50 ug/uL for Method Hot-shot, and 1 ug/uL to 10 ug/uL for Method Wizard SV Genomic DNA.
Something idea about How I can achieve product PCR+?.
I would be very grateful for his assistance.
The prblem could be with your dna. Check the od260/280 ration which should be close to 1.8. Only use 50ng dna per reaction. In case your primers are degraded get some dna from another researcher that amplifies in their hands to act as a pcr control. Start with an annealing temperature 8c below the lowest annealing temperature of your primers. Once you have a dirty amplification you can raise this temperature to get a cleaner product
I forgot to mention that if your primers include a sequence that does not exist in the template dna then you must not include that extra sequence when calculating the annealing temperature...only use that part of the primer that can aneal exactly to the template sequence
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