Hello, thank-you for reading this message first of all.
For the past two months, I have been trying to clone my insert 3.9kb fragment (cut with HindIII and AgeI) into pEGFP-N1.
1) PCR my fragment with 5'HindIII and 3'AgeI
2) Topo TA cloning
3) Sequenced - right size and sequence
4) Cut first with AgeI, gel purified, cut again with HindIII, gel purified
5) Ligate 1:1, 1:2, 1:5, 1:10 and 1:25 vector:insert at RT for 1hr (Roche) or 16C overnight (NEB)
6) Transformed into DH5a, XL-1 Blue, Top10
7) NO COLONIES!!!! OR COLONIES WITH NO INSERT!!!!
PLEASE HELP, THANK-YOU!!!
Cloning into GFP vector
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