Blunt end cloning

Irit Rappley via methods%40net.bio.net (by irappley from scripps.edu)
Wed Jul 6 20:36:04 EST 2011


If your problem is inserts with random deletions, have you double-checked that your PCRs and ligations are giving you correct sequences? You could try using a high-fidelity polymerase, if you don't already use one.

Do you think the deletions are happening upon expression and are due to the toxicity? If so, then you could try growing your E.coli at 30C or even 20C during (longer than usual) induction because the slower protein synthesis allows for better error correction. If not, then it seems to me that you have 2 separate issues (cloning the correct sequence, and subsequent toxicity upon expression). 

Why do you think that the problem is with your vector?

Irit


On Jul 6, 2011, at 4:57 PM, mnr mnr wrote:

> Hi Wo.
> 
> It is a outer membrane protein, toxicity is probably at expression level. I
> get inserts with random deletion that I can not use for functional assays.
> 
> Before I try other expression systems, I want to try other E. coli vectors
> first. Am I going in the right direction?
> 
> 
> On Wed, Jul 6, 2011 at 10:49 PM, WS <novalidaddress from nurfuerspam.de> wrote:
> 
>> Hi Matt,
>> 
>> does that mean that you don't get any clone with an insert at all or
>> "just" no clone that has a correct insert (full length, no deletions,
>> no frameshifts etc.)?
>> pUC18 is promoterless, right?
>> 
>> As you suspect inherent toxicity, do you have any idea what kind of
>> toxicity it is? Will you need that toxic element or may you make it
>> "innocent" by some sort of AA substitution / motif deletion? Can you
>> possibly switch to another host like some sort of phage, YAC, (really
>> safe!!!) virus etc?
>> 
>> Wo
>> 
>> 
>> 
>> 
>> On Jul 6, 7:23 pm, mnr mnr <mnr... from gmail.com> wrote:
>>> Hi all.
>>> 
>>> I have tried cloning a potential toxic gene about 1.2 kbp into E.coli
>> with
>>> T7 promoter. After two months, I am still not successful. I am now
>> plannig
>>> to do cloning into pUC18 vector before subcloning into few other vectors
>>> with different promoters. I have limited available restriction sites so
>> that
>>> I don't have to redesign PCR primers. Is it feasible to do blunt-end
>> cloning
>>> with Phusion polymerase into non-dephosphorylated vectors? Will I only be
>>> getting empty vectors after transformations?
>>> 
>>> Thank you.
>>> 
>>> Matt
>> 
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