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ligation problem although try everything,suggestions?

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ligation problem although try everything,suggestions?

Postby genetherapy » Wed May 16, 2012 3:31 pm

Hi all,
I'm in trouble with ligation nearly 5 months. My vector is 4775 bp. My insert is 500 bp. I also make PCR to obtain my insert (verify with agorose gel) and then I digested my insert after PCR with the enzymes which my insert have at it's ends (verify again with agarose gel) . Alternatively I cloned my insert to a cloning vector (did blunt end ligation with cloning vector, verify the ligation with agarose gel) then digested with the enzymes that my insert have at it's ends to get the insert from there. I digested my vector with the insert's same enzymes and make sticky(cohesive) end ligation. I used Quick ligase at room temperature(25 degrees) tried 5 minutes as the protocol said (NEB)alternatively try 25 minutes and also tried T4 DNA ligase at room temperature (NEB). I make 1:1, 1:3, 1:5 and 1:10 ligations. I make alkaline phosphatase and then try ligation again. I used 50 ng vector,15 ng insert it did not worked. I used 100 ng vector, it also not worked. I used invitrogen One shot top 10 competent e.coli. I tried competents cells with original vector in the kit. I used the S.O.C medium in the kit and also the S.O.C medium which I prepared myself. I add 250 mikroliter S.O.C medium after transformation. S.O.C diluetes the ligation,does it affect? I also tried spread transformants immediately without adding S.O.C. I get 2 colonies but they were the vector itself.
I really need help. Any suggestions will be appreciated.
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Postby JackBean » Wed May 16, 2012 4:19 pm

try O/N ligation at 17°C
http://www.biolib.cz/en/main/

Cis or trans? That's what matters.
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Re: ligation problem although try everything,suggestions?

Postby wbla3335 » Wed May 16, 2012 5:14 pm

I haven't done any lab work for many years, but in the old days, the buffers that came with ligases would often form a precipitate and needed to be vigorously vortexed before using. Have a look at your buffers to see if there's any precipitates. If so, try vortexing the **** out of them before use.
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Postby JackBean » Wed May 16, 2012 5:50 pm

Also, you may try to add fresh ATP to the ligase reaction
http://www.biolib.cz/en/main/

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Re: ligation problem although try everything,suggestions?

Postby genetherapy » Wed May 23, 2012 2:49 pm

I've bought the ligase 3 days ago. I was using NEB T4 DNA ligase.Now I'm using Quick ligase. I read at somewhere I can't remember now, it says do not spin ligase. Do anyone have experience like this. Can we centrifuge ligase I mean only spin 1 or 2 second? Does it effect it's activity?
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Re: ligation problem although try everything,suggestions?

Postby wbla3335 » Wed May 23, 2012 7:59 pm

Vortex the buffer, not the enzyme.
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Postby dustman » Thu May 24, 2012 10:42 am

You don't say what you do after restriction. Do you remove restriction enzymes? Or simply kill restrictazes by heat-shocking? Do you treat digested vector with CIAP (phosphotase)?

If you simply heat-shock restriction reaction and proceed with ligation, it can cause your problem. Several enzymes, like KpnI from NEB, aren't de-activated this way.

If you don't have 2-3 nucleotides added to your restriction site in primers, it could lead to problems with inefficient digestion as well.

These are just two but possibilities are many. Since you tried several ligases, problem might be not with them but at some prior step.
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Postby dustman » Thu May 24, 2012 10:47 am

Btw, read transformation protocol carefully.

You incubate specifically treated bacs with your ligation reaction and heat shock exactly as stated in the protocol by Invitrogen. 30 s at 42C is not taken out of blue. SOC is used for cells to recover before they are exposed to antibiotics in agar. You can probably vary incubation time from 40 min to 1h 20 min (I did), but less than that is questionable. Assuming 20 min division time for E. coli, 1 h = 3 divisions. I bet this is important.
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