ligation of PCR products

sam simons simonssp at pfizer.com
Mon Oct 14 09:18:31 EST 1996


I always get oligos with 5'phosphates if I am directly cloning the 
oligo, however I have not found this necessary when using PCR. I 
generally clone into a TA vector of some sort directly from the PCR 
reaction with no kinasing necessary. I have also directly cloned from 
the PCR reaction by adding restriction sites on the ends and then 
digesting the PCR product with the appropriate enzyme. Two things to 
keep in mind, 1. If you are directly cloning by digestion/ligation be 
sure to add a GC clamp of the appropriate length (this info is 
obtainable from the NEB catalog). 2. I prefer to TA clone first as 
this allows me to check my PCR product by restriction analysis and 
sequencing. It also provides a more efficient template for restriction 
than the linear PCR product.Good Luck.

Sam



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