Constructing a new multiple cloning site (MCS) from standard oligos
Order oligos that will hybridize to form the MCS and appropriate sticky ends.
Oligo hybridization
To hybridize phosphorylated oligos, mix the two phosphorylation reactions and bring the mixture
to 95ºC. Hold at 95ºC for 2 minutes and allow to slowly cool on benchtop.
Ligation to vector
For ligation, dilute hybridized oligos 50-fold into 1X NEBuffer 2; use 2µl of this insert preparation
in a 15-µl ligation reaction with standard amount of vector.