1 reply to this topic

[jonathannimal]

I have used two primers Forward and reverse to amplify my PCR fragment. I  have used two restriction sites SalI (in the forward primer) and BamHI (in the reverse primer).

There are the primers:

Cry4BSF: 5’- GGTCGACGTTCATAGGAATCCGTATCA -3’ (27 Mer) - Forward

Cry4BBR: 5’–GGGATCCTCACTCGTTCATGCAAA-3’ (24 Mer) - Reverse

I am not able to clone the product. I saw in the discussion that  there needs to be addition of sokme bases. Please help me & tell me that what sort of bases need to be added.

Please help me I am in a urgent condition.

With regards
nimal

[blasko]

You should add plus 3 nucleotides next to the recognition enzyme site.
                                                                           Cleavage %          
                                                                         2 hr             20 hr  
In case of BamHI:  cgcGGATCCgcg       >90%            >90%


                SalI:     ACGCGTCGACgtcggc   10%             75%

by NEB.


Bye!
B.