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Blunt end ligation insert:vector ratio


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#1 Sundus

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Posted 28 February 2012 - 10:25 AM

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?

#2 HOYAJM

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Posted 28 February 2012 - 10:35 AM

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.




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