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Simple Ligation/cloning questions

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#1 tsisqan



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Posted 30 August 2004 - 11:35 AM


I'm attempting to do a blunt-end ligation of my insert (2.3kb) into a linearized pCMV vector (6.3kb).

The vector was linearized with BamH1, blunted with Klenow, and Dephosphorylated (SAP) before being gel purified from agarose and extracted with Qiagen's Gel Extraction kit.

The linear insert was also blunted with Klenow and gel purified.

The cloning has had some efficiency problems (resulting colonies consisting of empty vectors without ligated insert) and high background on the vector only with ligase control. We typically do not use PEG4000 (we've had success before without it), but might try it now.

I have two main questions:

Is there any specific quality of dNTPs that I need to have for the klenow reaction to be efficient? our Grad student said ours were somewhat "low quality" dNTPs that she used mostly for quick PCR and she wasn't sure if quality was an issue with the Klenow enzyme working efficiently. If so, what brand of dNTPS do you recommend?


What protocol do you guys use for your ligation incubation? Our Fermentas T4 ligase suggests that we just leave it at 22 degrees Celsius for an hour...we usually leave it overnight...
Does anyone have a specific thermalcycler program or protocol for maximizing the efficienty of the ligation?


#2 bitpas



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Posted 30 August 2004 - 02:25 PM

in such cases it helps just to dephophoylate your vektor again, obviously a lot of your vektor was not dephosphorylated.

In fact i just had the same situation, a lot of background in the vector only control, but strangely when i screened the colonies from the "real" plate i had quite a lot of positives (12 out of 40). So i think it is worth screening even when you have a high colony number in the autoligation-control before repeating everything...

Klenow-dNTP's: I dont think a specific quality is needed. We use Fermentas-dNTP's and they work always fine.
Anyway i never heard about differences in dNTP quality, either they work or they dont work at all. Generally, if you can do a PCR with'em, they should be fine.

T4-Ligase: The most efficient Time:Tempertureratio for T4-Ligase is 12-16h at 16C, eg. overnight is fine. In difficult templates (eg. big vectors/inserts) it helps to do even 4C over the weekend (At least for me.. :) )
Perform the reaction in PCR-Tubes in a therocycler.


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