blue colonies

tflanigan at molbiol.ox.ac.uk tflanigan at molbiol.ox.ac.uk
Fri Apr 28 11:34:38 EST 1995


In article <bedal001-2504951746160001 at byrd.biochem.duke.edu>, bedal001 at mc.duke.edu (Wendy Bedale) writes:
> In article <3njeac$dc8 at mark.ucdavis.edu>, ez022056 at bullwinkle.ucdavis.edu
> (Edward Wang) wrote:
> 
>> I am currently doing PCR cloning using the Promega pGem-T system.  
>> Everything was working fine, and I was getting several hundred : 5 White 
>> to blue ratio, using Stratagene's super competent (10^9 efficiency) XL1 
>> Blue cells.  Because of the high cost, I switched to Promega's JM109 
>> (10^8 efficiency).  Now, my plates have lots of blue colonies : very few 
>> white colonies.  Does anyone know any explaination for this?
>> Also, we are trying to od Nrothern blots with probes made from 
>> differentialy displaying fragments (DD-RTPCR), but so far with no luck 
>> (nothing shoing on the blot).  Has anyone else had any similar problems, 
>> and perhaps even the solution to the problems?  Thanks in advance.
>>
> 
> 
> Is this a TA cloning kit?  If so, maybe your vector has lost its T-tails
> over time- I know that can happen with the Invitrogen TA cloning vector.


Edward,
not sure about the blue/white colonies, however the Northern problem, if 
everything else is OK, may not be a problem at all: it's possible that your 
message is low abundance and not detectable by Northern blots.  If you 
subclone your bands and sequence them then you can decide if they look 
interesting and maybe try semi-quantitative RT-PCR.
Good luck,
Tom. 



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