Seeking to amplify 2 genes: one is 441bp and another one is 88bp. The problem is that the annealing temp of the primer is 72degree.and the GC content of both frw and rev primers are 59.6 and 47.5 respectively. When I am doing amplification I am getting the 2 gene band separately.
Hi,
Its not clear wht do u mean by two genes? Are they continuous in frame and u have designed a primer set to cover both at the same time n get a common product?
In any case how big is the primer length? 72 is too high for PCR primer Tm. Max 23 bases primer set would work better. Adfding formamide to your PCR mix at 1% conc works for high GC content areas. so 1ul in a 10 ul reaction of Formamide.
Hope u can provide more details of ur work.
thanks for your reply....Actually both my genes are in continuous frame..i have attached my primer design for your view..
i have designed the primers to amplify both my AFP gene(441bp) and His linkers(88bp)
i have both the genes in the PCR amplification, but it was in a separate band...
the primer length is Frw - 52 and rew - 61.
waiting for your reply....
yes sebastin...the Tm of both the primers were very close to 72 or even higher then 72 (frw 81.2 (- 5) =76 and Rev 78.1 (-5) = 73.1)
the band are close to 88bp and 441 bp....
Hi, if in your technical data sheet (oprovided from the company) the Tm of both primers is near 72C..you should use as T of annealing a lower temperature ...so 67C about.
Bye
I must be a bit dull as I can't understand your primer design. Why have you added HIS tags on both the N and C ends which are 3' to your AFP binding sequence. Also your rev primer does not make sense.
Normally,
1) flanking sequences (ie those you want to introduce are done such that they do not interfere with the protein coding sequence (5' of ATG or 3' of stop)
2) Tm should be between 55 to 650C
3) The final 3 bases of your primer must be an exact match any mutations you wish to introduce cannot be within the last three basses (I know that's not what your doing here).
Work off the coding strand 5' to 3'
Forward primer : restrictions site/his tag-coding sequence
Reverse primer : coding sequence-restrictions site/his tag then reverse and complement.
Sorry Sebastian, have to disagree there. Though for the initial cycle, only the coding region of the primer will be complementary, in subsequent cycles the product of the previous round becomes the template and therefore the whole primer will anneal after cycle 1
Hi,
i looked at your primers. Do you want to fuse a 6 histidine tag to your protein of interest? If this is the case, why do you put the sequence of the tag at both the 5' end and the 3' end of your PCR product? If your objective is to tag your protein at the Nterm, the 6his encoding sequence you added in your frw primer is not at the good position. If you want to tag your protein at the Nterm, you need to put the CAT CAC CAT CAC CAT CAC just after the ATG (start codon) of your protein and then to continue with the 2nd codon of your open reading frame. The length of the sequence of your ORF added after the 6 his encoding sequence should be at least 20 nt to allow a good annealing with your target DNA. As it is designed your frw primer cannot hybridize with your target DNA as the 3' end does not anneal with your sequence.
Hope this will help you
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