Can someone help me on my primer design? (contains flag tag) - (May/19/2016 )
5’ ATG GACTACAAAGACGATGACGACAAG GCCGGGTGGAAC 3’ (Forward)
5’ TCAGTACTGGGAACG 3’ (Reverse)
FLAG GAC TAC AAA GAC GAT GAC GAC AAG
After PCR im putting it in a T vector so i didn't include any restriction enzymes. I'm also adding a FLAG tag to my forward primer. But what I'm worried about is the difference in melting point. Is it ok to just put random sequences in front of the reverse primer to balance the two?
First time making one ..thanks
It is better for the forward and reverse primers have similar length to avoid big difference in melting point. You need not to put random sequences for the reverse primer, just extend your primer length according to your original sequence.
Not quite right - the critical thing with the tagged primers is to get the gene specific sequences to have the same or similar Tm, you only need to include the specific part in the Tm calculations. If you use the full length, the Tm will be so high you won't be able to do the PCR.
This is so that the initial rounds of the PCR incorporate the primers specifically with the tag appended, this then fills in and is then used as template for subsequent rounds.