How to increase the efficacy of ligation when using single RE digestion - (Sep/26/2005 )

if you are worried about the double digestion of the insert then you should gel-purify it away from the old vector, only the inserts that have been cut with both Xho and Sal will be seperated from the vector.... that should save any problems with incomplete digestion...

HTH
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Well, i asked something that is not that obvoius... sorry maybe I should rephrase my question... let me put it this way: SalI has been know for its pretty bad digecstive capabilities. Alway, when using Sal I in my cloning strategies i notice that any time I cleave a plasmid with Sal I, the concentration of insert I get after gel isolation is always quite low. Are there any tricks now that could render SalI do its job properly?

According to New England Bioabs (my first stop for info on difficult restriction enzymes), it appears that Sal I is very picky about the buffer it finds itself in:

Activity in NEBuffers:
NEBuffer 1: 0%
NEBuffer 2: 0%
NEBuffer 3: 100%
NEBuffer 4: 0%


I don't think I've ever come across an enzyme that was 100% in one buffer, and 0% in all others...

They also include this note: "When using a buffer other than the optimal (supplied) NEBuffer, it may be necessary to add more enzyme to achieve complete digestion."

It sounds like you have incomplete digestion occurring. NEB also notes: "When cleaving close to the end of DNA fragments, cleavage should be done at 37°C for 1 hour using 10 units/µg of DNA with a minimum of 3 bases on each side of the recognition sequence." In your double digests (of vector, for example), are there sufficient bases 3' and 5' of the Sal I site to allow it to cut?

NEB further recommends you add 100 μg/ml BSA to the digest. More info here...

Good luck! Let us know if you can overcome your restriction problems -- for the Googlebot if nothing else!

Hi,
SalI is really the problematic enzyme and presently I am the next victim. Any suggestions?
Thanks in advance!

Hi every body,
I have clone a 3.2kb PCR fragment into pGEM-T and then cut out EcoRI (2.7kb) fragment and want to clone into a pBS in which I have already inserted 1.2kb fragment, I want to make a targeting construct. First of all I treated the vector with CIAP and got a lot of background, so then I change the CIAP now I did again now there are no colonies on empty vector plate, but I am also getting nothing on my target plate, I tried with different amounts of V:I like 1:3, 1:5, 1:10, but still its not working, the competent cells are good, the restriction enzyme EcoRI is there, I check both vector and insert, but still I don't know what to do now, any body who can solve my problem,
Thanking in anticipation,
Hammad, Vienna

Hallo, I did something similar, you should use more T4 ligase units(5-10) and ligate a big amount of the insert(2ug) vector(0.5-1ug) to get fragment ligated on the end XhoI then run ligation mixture on gel electrophoresis with ladder, cut fragment corresponding lenght of linearized vector and insert, then you should fill ends by Klenow fragment and ligate blunt end again



This is multistep way should work

Another way is to fill ends and ligate XhoI linkers to the blunt insert, then you digest like this modified fragment by XhoI and you have XhoI ends on both ends of the insert
good luck