Any experience with TOPO PCR cloning kit?

Bill McKendree mckendre at ASRR.ARSUSDA.GOV
Fri Jan 9 08:20:15 EST 1998


Yoram,  

I tried it once out of a kit my colleague purchased, and it worked 
suprisingly well.  5 min ligation using one microliter of a 50 microliter 
PCR reaction gave about 200 colonies 60:40 white:blue. Screening showed 
80% recombination.  I'm waiting for sequencing data now, but these 
results are similar to what my colleague has found, and his first 
cloning was successful.  

I might add that the PCR reaction I used was from degenerate oligos, and 
the products were rather small, which this method clones preferentially.  
However,I haven't has results from shotgun ligations this good since 1986.

Good luck,

Bill McKendree
USDA-ARS
Orlando, FL


On 9 Jan 1998, Yoram Gerchman wrote:

> Any one had experience with TOPO PCR cloning kit? How many colony can one
> expect and what yield?
> 
> .........................................................................
> Yoram Gerchman
> Division of Microbial & Molecular Ecology
> The Institute of Life Sciences
> Hebrew University of Jerusalem
> Givat Ram, Jerusalem 91904, Israel
> Tel: 972-2-6585175
> 
> 
> 




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