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5 replies to this topic

#1 sagar

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Posted 20 April 2009 - 09:53 AM

hi all

im trying to perform one ligation reaction for over 4 months and till now im not into anything.
i have a vector of size 2.8kb that is cut with ecori and hindiii and insert of size 1.4kb cut with the same enzymes(fermentas).i m confirmed about complete digestion taking place but the problem is with the ligation i have performed ligation using molar ratios of insert:vector in 1:1,3:1,4:1,6:1..............ihave tried all concntrations of vector startin from 50ng -500ng.

i have tried to dephosphorylate the vector using sap(promega).i keep it for 15mins in 37C and then deactivate the enzyme in 65C for 15mins.
Even i follow the small tips mentioned for ligation with sticky ends like heating the vector and insert in 37C before liagtion.
my ligation conditions are 16C for 16 hrs,4C for 12 hrs.
.
But I donot get any colonies- positive ones.My competent cells are good and they work properly.
Plz can anyone help me solve this problem.im going mad with this ligation .

#2 mastermi

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Posted 20 April 2009 - 12:15 PM

I do sticky end ligations always for just one hour at room temperature!
Otherwise strange things like a EcoRI HindIII ligation may occur (I already had this event twice!)...

#3 swanny

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Posted 20 April 2009 - 08:50 PM

Are you certain that the insert ends have digested completely? Are you sure the ligase is working properly?

Test both by doing a ligation reaction. Ligate insert to itself for ~20 minutes at RT, as well as DNA ladder. run a gel. If the insert and the DNA ladder don't shift upwards to larger bands, your ligase isn't working properly. If the size marker does shift but you only get monomer and dimer-sized insert bands, one of the enzymes hasn't cut. You need to have a multimeric ladder of insert to say both ends are digested and can religate.
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#4 Wonyong

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Posted 20 April 2009 - 10:07 PM

It must be a rare case but I once had a problem with cloning like you.

It turned out that one of the restriction enzymes I used was contaminated by exonuclease. The digested end of DNA was damaged and therefore no ligation could occur.

Try restriction enzymes from different stock.

Edited by Wonyong, 20 April 2009 - 10:08 PM.


#5 sagar

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Posted 21 April 2009 - 05:10 AM

Are you certain that the insert ends have digested completely? Are you sure the ligase is working properly?

Test both by doing a ligation reaction. Ligate insert to itself for ~20 minutes at RT, as well as DNA ladder. run a gel. If the insert and the DNA ladder don't shift upwards to larger bands, your ligase isn't working properly. If the size marker does shift but you only get monomer and dimer-sized insert bands, one of the enzymes hasn't cut. You need to have a multimeric ladder of insert to say both ends are digested and can religate.

hey thanks
i shall try ur suggestions and im changing the enzyme from a new company and fresh stock.

#6 Qundo12

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Posted 21 April 2009 - 04:21 PM

One more possibility is you got mistake in choosing RE for cloning. For example, your fragment has EcoRI and HindIII somewhere near the ends, so after digestion, actually it becomes the fragment with both ends are similar (EcoRI or HindIII), so this is no way to finish the cloning. Even it's the tiny thing but I had that some years ago due to careless designing. One more thing is you can increase the number of your sample in screening (colony PCR) by testing 4-5 colonies in the same tube (for 100 colonies you just need 20 tubes), this can increase your change of getting the correct one, very helpful in case of tough ligation.




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