Protocol Online logo
Top : New Forum Archives (2009-): : Molecular Cloning

Neeed Help , cloning Confirmation ?! - (Jul/12/2013 )

Hi All , i'm very happy to be connected to this forum :)
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 !!!

-Mohamed Rasheed-

Have you tried using primers which are based in the plasmid (e.g. sequencing primers)? Also note, I don't see a snab1 site in the multiple cloning site, are you sure this is the right enzyme to be using?


Thanks Bob for your Reply , yes i have tried my gene forward primer and plasmid sequencing reverse primer and recently i got 2 bands one of them at the specific size of my gene of interest , yes i know that SnabI is not present in the MCS however it's site is found upstream of the polyhedrin promoter of this transfer vector since my work depends on restriction deletion of the polyhedrin and replacing it with anthor one to make a compartive study on protein expression level , also i'am working on Synthetic gene cassette not a PCR amplified product

-Mohamed Rasheed-

Have you ran your recombinant plasmid (uncut) next to uncut unrecombined pFastBac1? Is the band larger?


That was also my problems.
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.


DrLeo on Mon Jul 22 09:08:42 2013 said:

That was also my problems.
I used PCR with specific primers of my inserts to check the cloning result from colonies of bacteria,

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.