No PCR Product for Sequencing - (Dec/21/2004 )
I have been trying to PCR exons 2,4 and 6 of the MTAP gene for sequencing purposes. I am using primers, the details of which I obtained from a previous study. However, I am not getting any products. I use 8ul of a master mix, with 1ul of DNA and 0.5ul of each of the primer pairs. The annealing temperature, I have set to Tm minus 5.
Could any one suggest any probable ways to troubleshooting, and getting a PCR products. I can provide further information about the procedure if anyone interested wants.
Have you checked the DNA after isolation: concentration, inhibitors? I suppose the primers are all right? The themocycler is all right? 8 µL mastermix sounds much to me, perhaps too much dNTPs or no Mg?
The DNA is highly concentrated, and worked previously for a GAPDH PCR. The primers should be all right, as they are not of my design. The study that used them previsouly had results with these primers. The mastermix worked with the GAPDH PCR, as well as with other PCRs performed by my colleagues in the lab.
Do you think it has something to do with the thermocycler, or probably the annealing temperature or time? The melting temp for both primers of exon 2 are 67.4 and 64.3 celcius. and the annealing temp that I use is 58 (Subtrating 5 from the lowest of the two). And the cycle is set to run for 30 secs. Is that where the error is?
30 s is not too long!? What amplicon size for? But what I meant was that the themocycler perhaps does not work properly? 58 °C sounds good to me, but you can perhaps retry with 50 °C to be shure.
I amn't sure about the working of the thermocyclers, I will have a word with my supervisor regarding them, as she herself once mentioned their proper functioning. However, considering they are working well, as they did for the GAPDH, what other errors might there be?
The size of the amplicons is 338 for exon 2, 271 for exon 4 and 328 for exon 6. Is that too much for the perimeters of the thermocycler programs.
You mentioned that 30 sec for annealing is less, so shall I make that a minute? Also, the primer elongation is run for 30 secs. Should I increase that time? The cyclers are programmed to run for 30 additional cycles, should that be increased?
I would honeslty appreciate your advice, as I cannot figure out why the procedure is not working for MTAP, whereas it did for GAPDH.
30s for annealing sound okay to me, but you should perhaps increase the elongation step up to at least 60 s.
Publications are fine things, perhaps there is someting wrong written? Check the sequences with the database?!