So I am performing cloning for the first time and i don't think I have done it correctly.
The sequence I want to clone is as follows. ATGGCCGGGCTGGGGGCGTCGCTCCACGTC...........GGATGCCAAGGAGAAACTGAGGATGTTCTC
I want to clone it into a pFastBac vector. between these two points:
ACTTTAAGAAAGGAAACAGCGATGCCTAGG///////CCTGCAGGACTAGAAGTACTATTTCAAGGA
I cut the vector with AvrII (CCTAGG) and PstI (CCTGCAGG), gel purified etc etc.
I used infusion cloning. I designed the following primers:
Forward: GAA ACA GCG ATG CCT AGG ATG GGG ACC GCC CGG
Reverse: TAG TAC TTC TAG TCC TGC AGG TCA CAT GAC TGT CTC CCA
The final complementary part of my reverse primer to the vector AGG should, in my mind align with the CCT?
However upon sequencing my the final sequence is (forward was fine) .......AACTGAGGATGTTCT CCTGCAGGACTAGAAGTA and right there between the TCT and CCT there should infact be 3 C's but I only have 2 and I have no idea why? any help would be greatly appreciated.