Efficient method for analysis of recombinants?


Thu Jan 30 09:38:26 EST 1997


MBP95GG at shef.ac.uk (G Garcia-asua) writes: > Rapid analysis of plasmids containing the insert can be done (according 
> to Maniatis) by resuspending the colonies in SDS, heating them and load 
> them into an agarose gel without ethidium bromide. After 
> electrophoresis the staining with EtBr should be done to visualise the 
> DNA and select the recombinants. 
> 
> My question is: does this actually work even with small inserts (i.e. 
> 300bp)?
> 
> Will Garcia-Asua
> mbp95gg at sheffield.ac.uk
> 


Will, I routinely use PCR to check for recombinants.  We have in our lab, a Idaho Technology 1600 Air Thermocycler.  
This machine allows us to do 30 cycles of PCR in about a half hour.  If your insert was generated by PCR you can 
re-PCR it up from a bacterial colony using the same PCR primers.  If it is not a PCR product you can PCR up the insert
region by using primers that are specific for the SP6 promoter and the T7 promoter or similar regions in your vector.  
You could also use primers that are complementary to your antibiotic resistance gene.  From colony to gel photograph, 
the whole procedure takes about 2 hours.  You could do the same with a heat block PCR machine but it would take 
considerably longer.  To prepare the template all you have to do is boil the bacterial colony in H2O for five minutes
and then pellet the debris.  A small aliquot of the supe is all you need for the template.  If you have questions, E-mail me, Mike.    



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