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Question Relating to LacZ Disruption


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#1 JamASharpe

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Posted 05 August 2013 - 09:02 AM

LacZ is used in blue white selection and contains a MCS.  The question is: is the MCS actually part of the LacZ gene or is the MCS a seperate piece of DNA stuck in the middle of the LacZ gene, but in frame with it?

 

I have just performed a 4 way ligation using both pUC19 and pGemT.  The vectors both had restriction sites added in by PCR (3' - MscI and 5' - XhoI) and the MCS was chopped out by this PCR.  The primers were designed to exactly chop out the MCS region and add in my RE sites precisely where the MCS was and leave the rest of the vector intact.  I now have lots of white colonies and only a very few blue ones.  Either my ligation and transformation has worked better than I could have ever hoped for or the LacZ gene has been completely disrupted by removal of the MCS and hence no blue colonies apart from the odd mutant.  If the ligation failed, I can't see how XhoI and MscI will ligate together and recircularise the vector - but then again, lots of strange unexpected things can happen in molecular biology so I guess there is a possibility of it doing so!

 

I will be doing colony PCR to check the status of my transformants, but in the mean time while I wait to get back into the lab I thought I'd pose the question.

 

Thanks in advance.

 



#2 JamASharpe

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Posted 06 August 2013 - 01:08 PM

Nobody seems to want to answer this, so I will post again.

 

The question is: is the MCS actually part of the LacZ gene or is the MCS a seperate piece of DNA stuck in the middle of the LacZ gene, but in frame with it?



#3 JamASharpe

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Posted 07 August 2013 - 02:00 AM

Colony PCR showed my inserts were correctly ligated into my vectors in all of my picks and hence the LacZ gene was functioning correctly.

 

Phew.



#4 phage434

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Posted 07 August 2013 - 06:07 AM

LacZ selection is (in my opinion) relatively unreliable and expensive. X-Gal and IPTG plates cost a lot, and the colonies often don't turn blue until the plate is left in the fridge for a day or more. If you insist on doing it, then I'd strongly suggest you shift to S-Gal, which produces a black colony, much more visible. But with strong antibiotic selection and backbone linearization, there is little need.



#5 JamASharpe

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Posted 07 August 2013 - 09:06 AM

Thanks once again Phage434.  My expression vectors (pET22b) which I'll be using if my sequences are correct don't have the LacZ gene and I had wondered how simple it would be to pick out positive clones.






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