Neeed Help , cloning Confirmation ?!
Posted 12 July 2013 - 01:43 PM
I have a question regarding cloning , i'm working on baculovirus expression system , my vector is Pfast-BAC-1 which is 4776 bp , my insert is 2236 bp
I have double digested my vector and my GOI With SnabI and HindIII , and ligated them and transform the ligation reaction into DH5alpha competent cells and got colonies on LB selective plates with Ampicllin
i have picked up the colonies and propagted them on lb broth with ampicillin and then i have carried out Plasmid miniprep to get my Recombinant construct , then i have checked cloning using gene specific forward and reverse primers and got a very strong band in gel electrophoresis at 2236 , when i have used gene specific forward and vector reverse primers it yield a multiple bands with band at specific size , so i try to make restriction analysis using the 2 enzymes used in cloning However it yield nothing (no cutting occurs) it give just a plasmid band ?!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!
Can anyone help me solving this problem , besides that internal primers for my gene yields a very strong postive bands !!!
Posted 13 July 2013 - 07:36 PM
Posted 14 July 2013 - 12:11 AM
Posted 14 July 2013 - 12:26 PM
Edited by Ahrenhase, 14 July 2013 - 12:26 PM.
Posted 22 July 2013 - 01:08 AM
I used PCR with specific primers of my inserts to check the cloning result from colonies of bacteria, I got strong bands. However, when performing enzyme restriction, there was no insert in my plasmid.
So, I usually use the blank-plasmid as negative control and I could see the same bands if my plasmid have no insert.
I think the reason is that primers could also bind non-specifically to the backbone plasmid.
Posted 22 July 2013 - 01:16 AM
This is usually because there is DNA from the transformation plated out on the plate, and then picked up when you are doing colony PCR.
That was also my problems.
I used PCR with specific primers of my inserts to check the cloning result from colonies of bacteria,
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