Sequencing

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S.Sandhya
S.Sandhya's picture
Sequencing

Sequence is cloned to p Bluescript vector and transformed to E.coli. Selected cloned aquired ampicillin resistant , but plamid is not visible in agarose gel. I wish to know what could be the reason? How to increase copies of plasmid?

SanDiablo
SanDiablo's picture
I need to know a little more

I need to know a little more detail about what you have actually done before I can help trouble shoot this. pBluescript is a relatively high copy number plasmid, so it seems unlikely that copy number is an issue - unless for some reason, your insert happens to be toxic to e.coli...

Did you perform the correct controls for your e.Coli transformation? It is possible you are looking at a contamination and not bacteria transformed by your plasmid of interest. (A "bugs only" control helps with this.) Are you sure you added ampicillin?

How are you checking for the presence of the plasmid? If you are trying to perform PCR directly on bacterial colonies, you will need to troubleshoot your PCR reaction, or consider growing mini-preps to check for plasmid.

If you have done mini-preps, trouble shoot the mini-prep protocol by including a control culture from something you know works. Check for the presence of DNA by spectrophotometry, then be sure you are using enough DNA so as to be visible in an agarose gel (at least 200 ng.)

Mini-preps have a reputation for poor quality DNA, so keep in mind that it may hamper your ability to perform PCR or a digest. If you are having trouble with mini-preps, consider going straight to Maxi preps. I think they are more reliable (especially when it comes to Qiagen.)

Write again when you figure it out and let us know!

Jason King
Jason King's picture
S.Sandhya wrote:Sequence is

S.Sandhya wrote:

Sequence is cloned to p Bluescript vector and transformed to E.coli. Selected cloned aquired ampicillin resistant , but plamid is not visible in agarose gel. I wish to know what could be the reason? How to increase copies of plasmid?

Following transformation, did you plate out the bacteria on LB-amp or just pour the transformation mixture in to LB broth? If you plated out - did you get well defined colonies on the plate or a lawn?

SD's point with forgetting the amp in the overnight culture sounds likely (who hasn't done that once....or twice?)

I had a similar problem once when I cloned the human elafin cDNA into pBluescript (and pUC) to do SDM on it. The problem was (i think) that elafin is known to have a bacteriocidal effect and thus there was heavy selection for clones that lacked the elafin and yes, there were clones that didn't even have the plasmid backbone either. Could your sequence be toxic?

I got round the problem by cloning the elafin cDNA into a pBluescript that already had coding regions on each side of the insertion RE site. This prevented expression of elafin from the T7 and T3 bacterial promoters.

There is one other possibility - but I'm sure it's not likely: EtBr in the gel?

COHscientist
COHscientist's picture
S.Sandhya wrote:Sequence is

S.Sandhya wrote:

Sequence is cloned to p Bluescript vector and transformed to E.coli. Selected cloned aquired ampicillin resistant , but plamid is not visible in agarose gel. I wish to know what could be the reason? How to increase copies of plasmid?

Sometimes, if you leave your plate at 37 incubator too long, there would be some satellite colonies and they don't actually have the plasmids in them.
That might be one reason.
Others, I agree with previous comments.