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Ligation not working....please HELP !!!!!

Ligation not working....please HELP !!!!! - Molecular Cloning Forum

Ligation not working....please HELP !!!!! - DNA cloning forum. Discuss the cloning of plasmids, vectors, DNA, RNA, cDNA, proteins, and other molecules for expression, sub-cloning, and other methods. Also discuss digestion, ligation, transformation, plasmid preparation, cDNA library screening, and competent cells here.


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Old 10-16-2012, 02:37 PM
Pipette Filler
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Default Ligation not working....please HELP !!!!!



I am out of my wits trying to get a ligation reaction to work. It would be very helpful if you could give some advise. The following are the conditions of the ligation reaction I am trying.

1) PCR amplification of template DNA (~3kb size) and gel purification using QIAquick purification kit.

2) Restriction digestion of pLenti6/V5 TOPO vector (~7kb size) with BamH1 and BstB1 for 1 hr at 37C. Gel purification of vector and hopefuly I did not overexpose the vector DNA to UV light while cutting the band from the gel.

3) Digested amplified template DNA with BAMH1 and BstB1 for 1 hr at 37C. Gel purified digested DNA with, what I think was, very little exposure to UV light.

3) Treated vector DNA with Antarctic phosphatase for 60 mins at 37C and then denatured enzyme at 65C for 8 mins.

4) Ligation reactions:

a) tried to ligate 58 ng vector + 75 ng template at 16C overnight

b) tried to ligate 58 ng vector + 75 ng template at 4C overnight

c) tried to ligate 58 ng vector + 100 ng template at 4C overnight

I did ligation reactions with all the above conditions to just test the ligation reactions on a gel to verify and saw a band at 10 kb and higher for ligation reactions and just vector and template bands in a control reaction without ligase.

I tried to transform stbl3 bacteria with the ligation reactions and I do not see any colonies. My positive control with circular vector plasmid was able to transform the bacteria but none of the ligated plasmids.

I am at my wits end trying to figure out what I am doing wrong.

Any advice will be be helpful at this stage.
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Old 10-22-2012, 08:57 PM
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Default Re: Ligation not working....please HELP !!!!!

Ah this ones easy! okay so the problem here is your digest BstB1 is most active at 65 C while BamHI is most active at 37 C. What you should do then is do 1 hour at 37 C then another hour at 65 C. This should fix your cloning issues my friend!
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