Cloning into GFP vector - (Jan/10/2005 )
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!!!
have you tried running your ligations out on a gel to see if it's being incorporated into the vector properly?
Hello! I'm also experiencing the same problem, except that my PCR fragment is 5'HindIII and 3'XhoI... I also have NO COLONIES, and I had run on an agarose gel where I saw a odd result: after the ligation reaction, the empty pEGFP showed a 12kb band and the pEGFP cointaining my 90pb insert showed a 9kb size. This is very strange, considering that this appeared also when I dephosphorilated the plasmid.
So, can anyone tell how the hell this is suppose to happen????