We have 3 plasmids with 10+ inserts each, that we are trying to propagate. As a convention, DH5a was used for cloning purposes of the first plasmid. The resultant plasmid was verified with insert specific sequences and the problem was that in addition to the target size bands, multiple other bands of greater and smaller sizes were seen.
next, we used BL21 as a cloning strain, and even though this is a strain used for protein expression, the issue of multiple bands was resolved.
This lead us to believe that bl21 was the better choice to clone these plasmids. But the actual problem rose when we tried to clone the rest of the 2 plasmids in bl21 and the same issue with multiple bands after PCR is observed.
What I want to know is,
1)why is this phenomenon occurring? is it related to the competent cells I am using?
2)If I use a fresh strain for making competent cells of either of the strains, can the issue be resolved?
1) If I understand correctly your are PCR amplifying your Region of interest from a whole plasmid or vector as a template. So, I believe that the extra bands you are observing are from the template plasmid or vector. Hence one thing you could do is in the agarose gel run your amplified region of interest in one lane and run your template plasmid or vector in the next corresponding lane. Use a DNA ladder to compare the sizes between the two. If you could see that both of them match then you need to perform Gel elution of your PCR amplified product if you are going to perform any further Downstream processing.
2) There is a chance that your PCR mixture (buffers, dNTP, Polymerase, Nuclease free water, MgCl2) is contaminated. How is your Non-Template Control? Do you See any Bands? If you see multiple bands in your non template control then your reagents are contaminated with DNA or plasmids.
Thank you so much for your reply. see there are further problems to both of your solutions
1) The plasmid I got has sequences pre-inserted into it, my task is just to amplify the plasmid. The problem is definitely with the vector I am using, as the PCR performed with the original plasmid gives single bands of the desired product size. My doubt is that the vector has something to do with the non-specific bands. I just don't know what? If known, the problem can be addressed
2) i observed a very light band in the no template control, but the band is of the exact product size as my desired product.
Are these multiple inserts homologous to each other so that you might be seeing recombination events that are changing the number of inserts in a plasmid? Although I would expect this to occur more frequently in BL21 since that strain is recombination proficient (and DH5 is deficient).
You might also just retransform your original plasmid stock (at low concentration) into DH5 again and see if the individual transformants are behaving better. It might not be a strain thing but it might be just that you did a clean transformation into BL21.
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