PCR Ligation Issues!!!

Zhonglin Chai Zhonglin.Chai at baker.edu.au
Tue Sep 27 19:31:30 EST 2005

Alternatively, you can directly clone your pcr products containing the extra A overhang into a T-vector, pGEM-T (Promega) for example. You do not need to treat or purify your pcr products for the cloning purpose using T-vector. After you confirm the insert you may then cut the insert out using the appropriate restriction enzymes (EcoRI and HindIII in this case) from the plasmid.


-----Original Message-----
From: methods-bounces at oat.bio.indiana.edu
[mailto:methods-bounces at oat.bio.indiana.edu]On Behalf Of ChenHA
Sent: Wednesday, 28 September 2005 2:51 AM
To: methods at magpie.bio.indiana.edu
Subject: Re: PCR Ligation Issues!!!

On 27 Sep 2005 08:09:29 -0700, "jssherkow" <jssherkow at gmail.com>

>Thanks for responding. I'm surprise anyone replied. Let me explain
>further. There are actually 2 clones I'm having trouble with. The first
>one is the PCR clone.
>I have a 503 bp PCR fragment digested on its ends with EcoR1 and
>HindIII. I do the PCR with Taq, which gives me overhangs. I then EtOH
>precipitate the PCR product and resuspend in 10 µM Tris-Cl pH 8.0 (the
>"T" in "TE"). I then "polish" the ends of the PCR with Vent polymerase
>(NEB) to fill in the overhangs.  I've even gone so far as to use some
>hot 32P dCTP to make sure the filling in works, 

Just a quick comment, it is unnecesary and pointless to do the
polishing step.   

> and I have gotten
>incorporation. After, I phenol cholorform and precipitate the product
>and resuspend again in 10 µM Tris-Cl and peform the HindIII reaction
>in NEB 2.

It might be worth just spending a few dollars on a kit (e.g. Qiagen,
GeneClean, etc.) for purifying PCR product and cleaning up after
enzymatic reaction.  Make life easier.

>  I EtOH precipitate that, resuspend, and perform the EcoR1
>reaction in its unique buffer. 

You should be able to do double digest with HindII and EcoRI.

> I phenol/precipitate that and resuspend
>in TE and run in on a purification gel (Crystal Violet, not ethidium
>bromide--works just fine). I cut out the band, electroelute it,
>precipitate it, and then I have my pure, terminal digested insert.
>I'm trying to clone this into pBluescript (pBSK+) which should be very
>easy. I perform the reactions exactly the same way.
>For the ligation I use 100 ng of (pBSK+) and a 3x molar ratio of my
>insert (0.153 pmol).  I perform the ligations the way I perform all of
>my ligations, which is overnight at 16oC.
>What's could possibly be going wrong? Thanks--Jake

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