I have 6 bacterial isolates, I have checked their band on agarose gel electrophoresis, I am getting a genomic band but when I am performing the PCR and after running the gel, I am not getting good results. I have tried several times still the same results... I have checked the purity of all 6 gDNA it is between 1.6 - 2, the primers I am using are IDT universal primers (27F and 1492R) with 1 microgram final concentration. Isolation I did use Promega extraction kit and PCR using Promega PCR mix I don't know where I am going wrong. Kindly help. I have attached gDNA and PCR images for reference
How much dna are you using in the pcr. It is very easy to use too much dna which may contain pcr inhibitors which can stop amplification. I would take one sample with a good OD260/280 ratio and run a pcr with dilutions of the dna something like 100ng, 50ng, 25ng, 12ng,6ng and 3ng using an annealing temperature that will certainly work with your primers....say 6c below the lower Ta of your primers and 35 cycles of pcr. Once you have a dirty amplification you can start raising temperatures to clean up the reaction but until you have some amplification the number of possibilities of why it is not working is very large
Try changing the concentration. There are some dimmers in left valves. Proper temperature is also important.
According to your electrophoresis, the 6 gDNA samples have different concentrations. Bring the DNA concentration to some optimal value, for example, focusing on the smallest of them. Then do PCR, for example, increasing the amount of micrograms of DNA, as in the third sample.
Do you have a good positive control DNA sample to include?
Paul Rutland is correct, it's easy to have too much DNA rather than not enough. I have my students dilute their DNA samples 1:10 & 1:100 & try PCR on all 3 concentrations (full-strength, 1:10, 1:100).
And double-check that no one has edited the thermocycler conditions. It's a common problem on shared equipment.
Good luck!
- Badges
- Science topic
- Similar topics
- Chemistry
- Pharmacology
- Pharmaceuticals
- Drugs