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Clean ligation controls but no insert

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#1 miST32



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Posted Yesterday, 10:23 PM

Hi all,

I've been working to clone several mutant alleles of the same gene into a new expression vector but have encountered a new problem (new to me, at least).  I have clean ligation controls and negligable background from the vector - However, despite getting between 100-200 colonies on my ligation plates and 4-8 on my vector + ligase plates, only one of my alleles has yielded a successful insert (verified by PCR and digest so far).  Pretty frustrating, but also fascinating - not sure what's going wrong.

I've designed the cloning strategy as follows:

Two REs:  KpnI (5' of insert) and XbaI (3' end of insert)  

1.  PCR amplify the vector (i.e. linearize it with primers containing RE sites and extra 5' bases, "in reverse" leaving RE+5'bases on the ends of the PCR product)
- No problem -
2.  PCR amplify the inserts with primers containing RE site and extra 5' bases.

- No problem -

3.  Check products on gel for size and purity (Vector is ~5100bp, Insert is ~3400, both are clean PCRs)
- No problem -
4.  DpnI digest the template away for 1hr.  (Might omit this in future attempts, but it has reduced the background).

5.  Purify PCR products with GuHCl and silica membrane spin column (QiaQuick system).  I do an extra wash with 35% GuHCl after buffer PB to remove potential leftover dimers and elute into warm EB.  Usually my concentration is 45-60ng/uL in 25uL elutions.
- No clear problem -
6.  Digest insert and vector independently with my two RE's for 2h at 37C in appropriate buffer (NEB 2.1) with 1X BSA.
- No problem, digests are complete in control experiments - 
7.  Ligate at 1:1 and 1:3 (vector:insert) for 2h at 25C in 10uL.  I use 50ng of vector and the aforementioned molar ratios of the insert.

8.  Transform full 10uL ligation into DH10b and grow overnight.

So far I've screened nearly 20 colonies from each of my dozen or so transformations across each of my alleles via PCR or digestion (or both).  As I mentioned, only one colony has yielded the correct insert!  

Usually I find that 10 colonies are more than sufficient to identify a correct insert with this method.  This protocol does work as indicated by the one positive clone, but the efficiency is apparently quite low - not good when I have multiple variants to clone.  

In my digest screening, I've noticed several uncut plasmid profiles migrating at different supercoiled/nicked sizes.  But again, they are not cut.  Both of my restriction sites are within the insert for this screen, indicating that nothing looking like the insert has been cloned into these pieces of DNA.  My first thought is that perhaps I've cloned my primers into the vector?  I am trying to avoid gel-purification since we only have UV in the lab, and I don't want to introduce mutations or further reduce efficiency.  Perhaps I should do it anyway?

Mainly, should I just play the numbers game and keep screening, or change the protocol significantly?

Thanks in advance!


#2 phage434



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Posted Today, 04:18 AM

You didn't mention a heat kill following your RE digestion. It is essential, to remove enzymes which, if still active, will cut your ligation product.

You can simplify your protocol by adding the DpnI digest to your other restriction enzyme digestions of the vector. 50 ng of vector is probably too much. Optimal for a 10 ul reaction is closer to 20 ng, which reduces concatamer formation relative to closed circular DNA.

You can likely reduce the template amount in your vector PCR to reduce background. You only need vanishingly small amounts as a template.

For your column cleanup, the real problems occur by inadequate washing of the column (do an extra PE wash). Then make very sure all of the ethanol is spun out of the column before eluting. Have you checked your DNA on a gel after cleanup?

#3 miST32



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Posted Today, 07:36 AM

Hi Phage434,

Thank you for the quick reply.

I will start the procedure over again today with your suggestions. KpnI is not heat-inactivatable, but XbaI is. I had not inactivated either, but this hasn't caused problems in the past. I will give it a shot. I hadn't thought that 50ng would be too much vector, so thanks for the tip there - I will try it later today.

To answer your question, I checked the purification last night on a 1% gel. The purified DNA runs clean for the insert, though there was a bit of high MW haze in the vector. Surprised by this because there isn't any such haze imediately following PCR. I presume this is some form of nonspecific product that is being enriched.


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