For the ligation reaction, the mass of insert can be calculated : (mass vector x size of insert in kilobase / size of vector in kilobase) x (insert:vector). How to then convert the ng into volume to withdraw from the pcr product ?
Hi there,
You need to determine the concentration of your sample either on gel by comparing band intensity with those of bands of the ladder which are known or by spectrophotometry (once you know the sample is pure).
You may use either an agarose gel to qualitatively determine the concentration of your vector and insert samples in comparison to a control.
A Nanodrop or spectrophotometer may also be used for precise determination if the sample is pure. This method will give you the DNA concentration in terms of ng/ul, which can then be used to calculate the volume needed per reaction.
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