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[waitingpooh]

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!!!

[vetticus3]

have you tried running your ligations out on a gel to see if it's being incorporated into the vector properly?