cloning kills - (May/22/2008 )
hi everyone,
i am facing a lot of trouble in handling cloning, hope you people can help out.
i hv been trying to insert a 1.1kb pcr product into IRES2-EGFP after removing CMV promoter from there. i cut my vector with aseI and Bgl II and gel purify the remaining 4.7kb fragment. for my insert i had aseI and BglII sites in primers. so, i gel purify my insert and digest with both enz. and do phenol-chloroform extraction as bglii cant be heat inactivated. i put ligation in 1:3 ratio n got good colonies. but the trouble is that all of them hv got no insert.
in fact when i recut with aseI and BglII to screen, i get a band size of 4.7kb and 600bp. which is similar to IRES pattern. but i had already purified 4.7kb part and put ligation. then from, where i am getting this pattern
ires egfp itself has a size of 5.3kb and i take out 4.7kb part after a dig of 12 hrs with aseI n bglII. my worry is that if i m taking full ires vector along with 4.7kb part coz wen i run digest i get two bands only 4.7big band and 600bp.
i hv been struggling for 3 months. please help me out.
thanks
Do you get colonies in your No-Insert ligation control? If so, there is probably uncut vector contaminating your linearized vector. The other thing you might consider is whether your restriction sites in your PCR fragment are far enough from the ends for efficient cutting.
dan
dan
yes, i do get even when i dephosphorylate the vector. even i think that probably uncut vector is contaminating my linearized product. can you tell me how to separate both coz i m worried if running a higher percentage gel can further create problem in gel elution or further processing. regarding rest sites i followed neb guidelines for putting excess bases for efficient cutting i suppose they are o.k.
thanks
dan
yes, i do get even when i dephosphorylate the vector. even i think that probably uncut vector is contaminating my linearized product. can you tell me how to separate both coz i m worried if running a higher percentage gel can further create problem in gel elution or further processing. regarding rest sites i followed neb guidelines for putting excess bases for efficient cutting i suppose they are o.k.
thanks
It's probably to late for any help now, but if not, here are a couple of suggestions.
1. Purify the linear vector after a single digestion (and do mock digestion to see if there are any co-migrating circular vectors). Then do the second digestion, and re-purify or heat inactivate second enzyme.
2. Another possibility is that the circular plasmid will not co-migrate with your band of interest (double-digested), if you do not include EtBr during electrophoresis (just stain after the run.
Also make sure the insert is in molar excess in the ligation rxns.
-d
It's probably to late for any help now, but if not, here are a couple of suggestions.
1. Purify the linear vector after a single digestion (and do mock digestion to see if there are any co-migrating circular vectors). Then do the second digestion, and re-purify or heat inactivate second enzyme.
2. Another possibility is that the circular plasmid will not co-migrate with your band of interest (double-digested), if you do not include EtBr during electrophoresis (just stain after the run.
Also make sure the insert is in molar excess in the ligation rxns.
-d
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THANKS i hv been waiting for your reply. can you please tell me what you mean by mock digestion?
if i misunderstood you, plz. correct me. do you mean that i shd. cut my vector with first asei and gel purify ( this will lead separation of uncut from single cut) after that i shd do cut with bglII and again gel purify (as i will reqd to remove 600bp fragment generating from double cut). But one doubt in last step, i have is that what if single digested fragment ( size5.3kb) and double digested fragment(4.7kb ) do not separate and i end up purifying both together.
A mock digest is simply not adding the enzymes to the reaction. Supercoiled DNA may nick at some rate, particularly if you are doing long digests. So, uncut DNA having been incubated at 37 with Mg may have a different ratio of supercoiled to nicked than your plasmid prep. This control is rarely needed, I just thought it might help you,
I think you understand me, but your last point is important (and could be the root of your problem). If one of your enzymes is not cutting efficiently that would give you the results you've seen too:
-your singly cut vector would easily recircularize, and your insert would still be blunt at one end.
of course, you'd expect to see a lot less colonies from your no-ligase control (not sure if you did).
Make sure you do single digests with both enzymes as a control too. If you are not getting a complete double digest of your vector, expect your insert to be equally incomplete. Pick a %agarose that best separates the 4.7 and 5.3 bands. Add phosphatase the last 15minutes of the reaction. No need to phenol extract, if you are gel purifying fragments.
good luck.