Control Restriction Digestion - got one additional band - what does this mean? (Jul/27/2009 )

hello,

i transformed my ligation-product into competent E.coli - when i check for the right colony i picked 10 colonys and did some prep and control digestion.

i checked with the 2 restriction enzymes i did my cloning with (hindIII & EcoRI)! according to the agarosegel i did afterwards i detected products with insert of the right size, but additionally i also get a product which is bigger than my insert.

could this be a kinda contamination??

what should i do, start again with the ligation?

thanks for any suggestion!!

moljul on Jul 27 2009, 07:06 AM said:

So you got your insert band, your vector band, and one additional band? If the additional band is larger than the vector, is it possible it's your plasmid digested with only one enzyme, i.e., a linear plasmid as a result of an incomplete digestion?

fishdoc on Jul 27 2009, 05:16 AM said:

thanks for reply,
the additional band (~1000bp) is between my vector band (~5000bp) and my insert (~200bp)! so incomplete digestion as a reason could be eliminated!

Any time I'm in a situation where my restriction digests don't match what bands I expect, I send the plasmid for sequencing using primers outside of where I cloned. In addition, I begin the process of re-cloning in the event that sequencing tells me there was a mistake.


Could you give a little more information as to how you obtained your insert? Was is a PCR product or a digestion product from another vector? If it was a PCR product, were there any non-specific bands, and if so did you gel purify or PCR purify? If it was a digest of a plasmid, did you gel purify? Is there a chance the digest to obtain your 200 bp product also generated 1 kb product from the plasmid, i.e., does EcoRI or HindIII cut more than once?

It could be uncut supercoiled form. Take less DNA and digest. Also, check with other enzymes.

Best,
TC

moljul on Jul 27 2009, 07:59 PM said:

fishdoc on Jul 27 2009, 07:36 AM said:

thanks fishdoc,
i want to produce some truncations of a protein i am already worked with! therefore insert was generated by PCR from vector with 2 specific primers (including restrictionsites)! i was able to get one nice band by PCR and gelpurified them to do restriction digestion afterwards. i also did some restriction/gelpurifications with my vector! purified products where ligated as recommended! i think i did all the steps correctly!?