First of all you need to identify where the start and stop codons are in those sequences... unless you want to clone the UTRs also.
If you want to clone the full sequence excluding UTRs, then you have no choice about where to place the primers, other than length... so your specific primers are the start 18-25 bp and the final 18-25 bp (reverse complemented) of your gene sequences. YOu can calculate the Tm by using the following formula: Tm= 4*(number of G and C) + 2*(number of A and T). You can adjust the length of the primers to compensate for differences in the sequence giving different annealing temperatures.
The annealing temperature you will use in the PCR is calculated based on the specific component only, ignoring the bits added as these don't play a part in the PCR initially.
Restriction sites and other add-ons go on the 5' end of the primer always.
You will probably want to add a kozak sequence (GCCACCATG, where the ATG is the start codon of your gene) to the 5' end if you are planning on expressing these in eukaryotic cells.
So your forward primer will look something like:
[Nde1site][kozaksequence][18-20bp of codingsequence]
Reverse will look like:
[Xho1site][reversecomplement of 18-20 bp of codingsequence]
FluffyMember Since 18 May 2011
Offline Last Active Feb 20 2013 01:42 PM