Blunt end ligation insert:vector ratio - (Feb/28/2012 )

Hey,

So my experiment goes something like this......Firstly, my gene of interest(575bp) was PCR amplified using phusion Taq . The band obtained, after the PCR product was run on 0.8% agarose gel was excised. With the help of gel extraction kit the DNA from the gel was eluted. The concentration of the eluted product obtained on the nanodrop was 13.6ng/ul.

In order to obtain efficient ligation of the PCR product with the 3.5kb blunt end vector what ratio of insert:vector should I use?

You can try different ratios for the ligation reaction. I usually start at a 3:1 vector:insert ratio and will try a 5:1 and 8:1. There really isnt a set method for determining the optimal ratio. Trial and error.