Question

Why is it necessary to find the new vector plasmid concentration after undergoing digestion and the clean up?

During the digest clean up what buffers are used and what is the purpose of each one?

Answer 1

Maintaining perfect vector insert ratio is an essential step for sucessful coloning of gene of interest. Normally for sucessful ligation in cloning experiments, vector & insert ration is maintained as 1:3 respectively. The vector and gene of interest (Insert) are isolated and purified (Clean-up using gel elution) and then subjected to restrction digestion using suitable restriction enzyme. The concentration of both is determined using nanodrop so that right volume of vector will be used to maintain the ratio as 1:3 (Vector & insert). However, if this vector-insert ratio is not maintained then chances of ligation and sucessful transformation/cloning are minimal.

The restriction digestion buffers are combination of cations and other components so as to maintain the suitable pH for the reaction to accomplish. The normal pH required for the restriction enzymes is 7.0–8.0 and the most common buffer used is Tris-HC. Depending upon whether single digestion or double digestion is performed, then a buffers are used singly or in combination depending upon the compatibility of buffers for enzyme activities in each buffer. The selection of specific buffers is very important so as to acheive optimal activities of both enzymes and also to avoid star activity.