subclone help - (Jul/07/2006 )

Hello every expert:

I am sruggling with my clone now and wanna find out if anyone can help. I amplified my insert (1kb) using PCR with BamH1 and ECoR1 designed at 5' and 3' end respectively and then cloned it into TA vector. Then I performed the colony PCR and minipreps and digested the inserted TA vectors with just BamH1 and the fragments of interest released from the TA suggesting my clone was successful. then I cut out the band and prepared pCDNA3.1c and pCDNA3.1 flag-tagged c vectors by digesting with BamH1 followed by dephosphoralytion. The product has also been genecleaned and subject to ligation. The ligation was performed using my insert with pCDNA3.1c and pCDNA3.1 flag-tagged c respectively and I did the plasmid only control. Then I transformed the ligation mixture into Top 10 competent cells.

The problem was there was no evident difference of the cology numbers between the control and recombinant ligation (the control has 20-30 less cologies than that of the recombinant and total colonies were about 80-100. Then I picked 24 of the colonies and did minipreps and digested them with BamH1 but no fragment released.

I also digested my minipreps with KpnI restriction enzyme. This would tell me the orientation of my insert which if the orientation is right, the fragment will be expected at about 700bp. The strange thing was all the 24 minipreps got the 700bp fragment released which was contradictory with the previous no expexted bands came up with BamH1 digestion. I repeated the process twice and the results were relatively the same.

Could anyone help me with this problem or give me some suggestions?

Many Thanks

I would suggest to digest your plasmid DNA again with other restriction enzymes. Maybe you chose on that cuts your vector and insert once and also would tell you the orientation of your insert.
I think your BamHI sites maybe cannot be cut any more after restriction digestion and ligation. That happens quite often.
Did you check the total size of your linearized plasmid-insert-construct? Is it larger than the linearized vector?

Thanks for your help Chalet2

But one thing I am just wondering is that why the BamH1 digestion will not be viable again after the first digestion. Because works done in my lab suggest that they can be re-digested. Can you explain why they are not be able to work again. The sticky end is reconstituted again after the ligation, they should be fine when you use the same emzyme to split them.......


Thanks