Dear colleagues, I would like to ask a question about primers and PCR protocol for the amplification of the Arginine Kinase gene in insects. We are trying to amplify a fragment of this gene using primers 183F / 939R in beetles (Elmidae, Dryopidae). We have tried several protocols with different annealing temperatures (see below) but the results are very poor. I would like to ask you to share your experience in amplifying ArgK in insects and recommend a functional protocol or another set of primers. Thank you in advance.
Primers used: 183F / 939R
Protocols used:
3min 94°C; 35x (30s 94°C – 30s 53°C – 60s 72°C); 10min 72°C
3min 94°C; 35x (30s 94°C – 30s 57°C – 60s 72°C); 10min 72°C
2 min 94ºC; 38x (30s 94ºC – 30s 57ºC – 120s 72ºC); 5 min 72ºC
Hi David
you should verify if your tax is a hot start one, meaning maybe you need to extend the first step.
in view of your programs, I would use a touch down PCR, starting at 57°c to 52°c, 0.5°c by cycle, for 10 cycles, followed by 30 cycles at 52°c.
you should also use DMSO at 5% to enhance efficiency, but first try an in silico PCR at NCBI to set the right Tm.
all the best
fred
Do you have a good positive control to test out?
Also, do you have primers for another gene that you know works very well in insects (e.g. actin gene) to make sure your DNA extractions were successful?
Good luck!
Are you amplifying gDNA or cDNA and is there an intron between the 2 primers which might make a very long amplimer if using gDNA?
I will advice you include a positive control as mentioned by @katie, to be sure your nucleic acid samples are good. If you h'v confirmed it's purity and potency and you get wake results, you then go for gradient PCR to optimize your pcr profile. I'm sure this is gonna give you a better profile for your amplification.
- Badges
- Science topic
- Similar topics
- Biological Science
- Molecular Biology