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PCR product now not getting with same primers and other PCR conditions - (Mar/21/2012 )

Hi,

My both forward and reverse primers (HPSF purity) are of 21 bp in length and having Tm 57.9 as per the manufacturer . Primer original stock is 100pmol/microliter and working stock is 10pmol/microliter. We used Fermentas 2x ready to use PCR master mix and primer concentration was 0.8 micromole. PCR conditions are as- initial denaturation: 95oC for 10 min, denaturation: 94 for 30sec, annealing: 50oC for 30 sec, extenstion: 72oC for 30sec, final extension: 72oC for 10 min. Total 35 cycles. My PCR product size is around 300bp. Initially I got sharp band at 300 bp alongwith thick primer dimers. Then on that basis I designed my cloning primers (for infusion cloning method of Clontech) from same manufacturer. While I did PCR with my cloning primers I got very faint band. So I did PCR by using both original and cloning primers I do not get band in both reactions. Sometimes I got faint band by using original primers but at the same time I do not get band by using cloning primers. I tried gradient from 46oC to 53oC for annealing. I also increased PCR cycles upto 45 then also there was no change. I made fresh working primer stock and also did fresh RNA extraction, cDNA synthesis and then PCR but still the problem exists. I also checked my primers online by using Netprimer site for dimer formation, hairpin formation etc. My primers have hairpin formation, dimer, palindrome sequence and also runs. What should I do so as to get sharp band again. please suggest me solution for the same.
Thanks

-ajitk76-

I would do a gradient from 48-63 in annealing temperature. What is the GC content of your template? The extra cycles will do nothing except cause problems. It sounds as if a redesign of the primers may be necessary if you have strong hairpins and dimers. I don't think purified oligos are necessary for this.

-phage434-

phage434 on Wed Mar 21 12:26:28 2012 said:


I would do a gradient from 48-63 in annealing temperature. What is the GC content of your template? The extra cycles will do nothing except cause problems. It sounds as if a redesign of the primers may be necessary if you have strong hairpins and dimers. I don't think purified oligos are necessary for this.


GC content is 47.6%. How should annealing temp. is greater than Tm. as you say.

-ajitk76-

Because Tm is poorly calculated, in general, and I don't trust the typical computations at all.

-phage434-

I am still thinking what are the reasons for getting faint band or not at all when initially there was sharp band with same PCR conditions. My other primers also have primer dimer and some also hair pin but they worked well in PCR getting sharp band. so what are the possible reasons for the above problem and solutions for the same. I have done sequencing of my gene when I got sharp band initially, so should I synthesize new primer set on the basis of sequence? or should I look for other primers in literature? I also blast my sequence in NCBI primer blast to get specific primers for my gene but in the blast result the size of my PCR product is showing less (around 216 bp) instead of around 300 initially. Is there any problem with sufficient production of protein after gene expression in cells (in vivo and in vitro)? I want to clone together 4 different genes of around 300 bp from 4 different serotypes of organism. Is this much reduction in PCR product is OK ?as my other PCR products are of around 300 bp and I want to clone them together. Thanks

-ajitk76-