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I canīt obtain the amount of DNA required for ligation of blunt ends

Ligation phenol extraction Klenow CIAP

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#1 manueliux_06

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Posted 24 April 2012 - 11:25 AM

Hi, my english is not so good, I hope can explain correctly.

I'm trying to subclone expression cassette (3392 bp) of pHANNIBAL to pCAMBIA. I make digestions to pHANNIBAL with Not I to liberate the insert and pCAMBIA with EcoR I to to linearize the vector approximately 3 micrograms of DNA. After the digested mixture is added Klenow for fill-in reaction according indications of invitrogen which found that terminate fill-in reaction by phenol extraction.


And here is the problem because after the phenol extraction I quantified in NanoDrop and only get 1-2 ng /ul. This amount is not sufficient to continue the dephosphorylation with CIAP. Previously I cloned with cohesive ends and added CIAP to the digested mixture for dephosphorylate, then I run agarose gel and I purified bands with kit of General Electric. Finally I obtained (10-15 ng / ul) After I make ligation reaction and the cloning fragment is done, but now, I'm stuck with using Klenow because after phenol extraction I have little DNA.

#2 chandra3316

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Posted 03 November 2013 - 03:52 AM

Hi! Manueliux_60,

Did you solved your problem. Because I am also facing similar problem in my cloning expriment. could you please let me know any interesting trouble shooted points we must taken care(while doing purification/etc) to increase yield.?



#3 HOYAJM

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Posted 06 November 2013 - 09:26 AM

You are obviously losing the majority of your sample in the digest/fill-in/extraction. The simplest solution is start with a much higher amount of vector (~10ug). I have observed similar problems when doing a fill-in reaction after a digest. I gel purify my samples instead of phenol extraction for what is worth.

 

What I find really saves time and effort is finding a way to do sticky end cloning. Just make sure this is not an option before continuing.







Also tagged with one or more of these keywords: Ligation, phenol extraction, Klenow, CIAP

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