miniprep substitute

Jeff Huckaby huckabyj at pantheon.yale.edu
Thu Sep 18 13:28:36 EST 1997


We routinely use "colony PCR" to screen for inserts in high copy numbers
plasmids...if your primers do not amplify the host cell DNA, then you can
do the following.

Set up your PCR reaction (we use 20ul)
Use a 20ul tip to pick up a colony.
Resuspend colony in PCR reaction.
Drop 20ul tip into 1 ml of LB if clone may be needed for future use
Do a 95C inc for 2min
Amplify...
This works for use >90% of the time...ocassionally we miss inserts...
If we need the clone for future use, we add 2 mL of LB amp culture to the
1 mL culture for an overnight and subsequent miniprep...
Jeff Huckaby

==============================================================================
Jeff Huckaby                                            Jeff Huckaby
Department of Biology					426 OML
email huckabyj at minerva.cis.yale.edu                     Yale University
office:(203) 432-3877               		  	165 Prospect St.
WWW http://pantheon.yale.edu/~huckabyj/topbio.html	New Haven, CT 06511
Lab:(203) 432-3892					
==============================================================================



On Thu, 18 Sep 1997, Peter Schuchert wrote:

> I heard that colonies of plasmid transformed E. coli
> can be examined for cloned inserts by boiling a little bit
> of the cells and using this for a PCR. 
> Could anyone please give me more details on this protocol.
> Thanks in advance,
> Peter
> 
> 
> -------------------------------------------------------
> P. Schuchert
> University Hospital INSEL
> Dept. Gastroenterology,  MEM-B814
> CH-3010 Bern Switzerland
> Tel 0041 31 632 84 92    Fax 0041 31 632 96 65
> http://www.unibas.ch/dib/zoologie/research/schuch.html
> -------------------------------------------------------
> 
> 


==============================================================================
Jeff Huckaby                                            Jeff Huckaby
Department of Biology					426 OML
email huckabyj at minerva.cis.yale.edu                     Yale University
office:(203) 432-3877               		  	165 Prospect St.
WWW http://pantheon.yale.edu/~huckabyj/topbio.html	New Haven, CT 06511
Lab:(203) 432-3892					
==============================================================================





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