Hi,
I am experiencing some problems with my ligation. I amplify my gene of interests from Enterococcus (~1000bp) + restriction sites for BamHI and SacI + thrombinase sequence and His-tag. Cut it with the 2 enzymes (FD) and purify via gel purification (making sure it is the right size). Then I take my plasmid cut it with the same restriction enzymes, dephosporylate and gel purify and ligate over night ~1:2. Transform in electrocompetent S. aureus. The few clones I get have always the same false insert (~150 bp) + Histag and thrombinase + restriction sites, for all different genes I tried to insert.Sequencing shows that the insert stems from Enterococcus.
It can't be false amplification. This has to be excluded after gel purification.
Perhaps try to create the plasmid first in a E. Coli strain which is used for cloning (i.e. recombinase deficient, etc.), amplify your plasmid there and then use it for transforming your S. Aureus.
During ligations you are working with minute amounts of correctly ligated plasmids, then only a fraction of the bacteria takes up the DNA and under selection pressure they will just your gene carrying the resistance marker and get rid of the rest of useless DNA. If you start with a lot of "correct" plasmid DNA, your chances are higher that one clone will be "correct".
Hi Ann-Kristin,
It sounds like you might be having some issues during the preparation of your insert/plasmid and maybe even ligation. Just to clarify, are you gel purifying your insert prior to cutting with your RE's? It is important (sometimes) to remove the PCR buffer prior to cutting with enzymes. Some enzymes work wonderfully in standard PCR buffer, but some do not. BamHI and SacI have different buffers so make sure you are digesting appropriately. The same goes for preparing your plasmid.
I would recommend amplifying your insert and gel purifying. Then, perform a single digest on your insert. Perform the same digest on your vector then gel purify both. Repeat the process with your other enzyme. Once you have your purified products, CiP treat your vector and either gel purify again or heat inactivate and then you can proceed with your ligation.
Check your ligase....is it old? Does the buffer require the addition of ATP or is that supplied in the buffer? Also, I would start with a 1:3 molar ratio of vector to insert and if that doesn't work, you can alter the ratio appropriately. If you have the ability, maybe try a chemical transformation instead of elecroporation.
Good luck!
Thanks for your suggestions. I purify after digestion. Might be that restriction is not working properly with PCR buffer. I use fast digest with corresponding buffer in which double digest should be working.
Though the vector i am using is linearized (with same restriction enzymes), but after ligation another fragment is inserted. Do you have any idea for that?
Are you sure that your S. Aureus strain is suitable for cloning?
Do you have some extra bases at the 3' end of your primers?
Hi, yes I do. It was used before with the same plasmid, but with genes from S.aureus and not Enterococcus.
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