I am going to use restriction digestion to clone my PCR product (1.6Kb) into the expression vector. I have to design a forward primer with HindIII restriction site. I went through the NEB website which says minimum 3bp can be added before the HindIII site. Can anybody give me designing suggestions for this primer?
For example: CACAAAGCTTyoursequence should work.
Don't forget that for melting temperature only "yoursequence" matters.
Make sure you add a few base pairs at the end for close to ends digestions (New England Biolabs has an assessment of this issue on their web site). I always make my terminal nucleotide a G or C. Definitely make sure you use a proofreading polymerase too. I've used a number of different ones with success but if your target fragment is GC rich, GCAccuprime from Life Tech works well.
i will be using accuzyme mix for PCR by bioline. hope that works!!
and also will the overhang affect the annealing temperature??
Farkas remark is spot on- use the annealing temperature for your initial sequence (w/o overhangs). It is what you need for the initial priming for the most efficient and specific amplification of your target. Once you get into exponential amplification of your target, your product will be in terrific abundance and priming at the same annealing temp will be no problem.
Hi Apoorva,
If you are not already using a specific software to do that I would recommend to use Clone manager. It allows to design your primers from your template sequence and will tell you if you have any issues with false priming, and you will also be able to simulate PCR with your primers and template and get the possible products at the set conditions.
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