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Cloning into GFP vector


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#1 waitingpooh

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Posted 10 January 2005 - 02:51 PM

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!!!

#2 vetticus3

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Posted 10 January 2005 - 06:26 PM

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




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