PCR Conditions for primers with GC Clamp and without GC Clamp - (Jan/19/2007 )
I did a touchdown PCR for my DNA extract using primers 341f-GC and 518 r. After running the DGGE gel, I extracted my bands of interest, store them in 40 microliter of distilled water overnight and re-PCR using 341f and 518 GC using the same PCR conditions for sequencing. Is it a right procedure? Or do I have to vary my PCR conditions in the primer without GC clamp? Does the presence of GC clamp in the primer affects its melting temperature?
Hoping for your answers...
there maybe a difference in but I have not noticed.
And just to clarify matters, the seqencing cycle is different from the PCR amplification cycle
The cycle conditions that I use (with big dye terminator 3.1)
Melting = 96 C (15 sec)
Annealing = (tm - 5 C )(20 sec)
Extention = 60 C (4min)
No of cycles :25
The Annealing temperature is the tm of the individual primer minus 5 Celsius. Unless you have standarised the tm of all your primers, the annealing temperature should follow the tm of the specific primer in use.
Each primer is individual so there will be slight differences but if the annealing temperaure is similar then I would use the same conditions. The GC clamp will affect annealing but you still have another 20 bases on the primer and they're going to have the major influence.
What would happen if I use the same annealing temperature in PCR for primers with different annealing temperature? Would this result to mispriming? Actualy, I did a DGGE analysis using a 341-f with GC clamp and 518 r and then excised the bands of interest and did another PCR using 341-f and 518 r using the same annealing temperature with the previous one with GC clamp. I think I got the desired product size based on the gel electrophoresis results. One of my problems that I encountered however was that the results of the sequencing showed that the bands that are located in the diiferent positions in the DGGE gel have the same sequence? Could that be the result of mispriming?