SubCloning

Digest Vector and Parent plasmid DNA at 1:3 ratio with restriction enzymes

use 15 ul reaction

for 30 min at 37 oC

Run on agarose gel. Must use fresh 0.5 X TAE buffer. Make sure wash gel tank with ddH2O

Cut out insert and vector.

Put insert and vector gel slice into the same 1.5 ml tube.

Add 1 ml QG buffer

incubate at 55 oC for 10 until gel slices are completely dissolved

Transfer liquid to a column, Spin for 30 s

Wash with 2 X 0.35 ml of PE buffer, spin 30 sec

Spin for another min at max speed

Add 25 ul ddH2O, spin for 1 min at max  speed.

Add 2.5 ul 10 X ligation buffer (must completely thawed and mixed by vortexing)

Add 1 ul T4 ligase

Ligate at room temperature for 1 hour

Add 100 ul NEB Turbo or DH5alpha

Incubate on ice for 20 min

Heat shock for 60 sec at 42 oC

Incubate on ice for 5 min

Add 1 ml SOC

Culture for 30 min to 1 hour at 37 oC in a shaker-incubator.

Spin down cells, dump liquid, leave only ~100 ul medium

Suspend cells and Spread on LB plate with antibiotics.

Grow at 37 oC o/n

Colony screening and restriction digestion, and sequencing if required.

Document all experiment details on ELN