BISC311:Cloning

Step 6 Cloning: for RNAi, use Topo vector Materials: 	LB plates LB broth Ampilcilin Qiagen miniprep •	Place 4 plates with lids open at 37C

2 tubes DNA 	0.5	4 Salt solution 	1	1 Sterile water 	3.5	0 TOPO vector	1	1 TOTAL	6	6

•	30 minutes at RT •	add 2ul to -80C vial of One Shot Chemically competent E coli (thaw on ice) •	Incubate on ice for 30min •	Spread 30ul of Amp (50mg/ml) on plates •	HS at 42C for 30s •	2 min on ice •	add 250ul of SOC •	shake for 1 hr •	spread 100 ul of transformation on plates. Spread and leave at 37C over night

Pick colony •	5 colonies picked and grown in 2 ml of LB+2ul of Amp in culture tube. Culture for at least 24 hrs at 37C with 250rpm shaking.

Plasmid isolation (follow protocol manufacturer’s protocol) For the Qiagen kit, here is a brief summary: •	Place 1.5ml of the culture into 1.5ml tubes and spin for 1 min. Use QIAprep Spin Miniprep Kit to purify the plasmid. •	Resuspend in Buffer P1. Pipette up and down till homogeneous •	250ul of P2 added. Mix by inverting 4-6 times. < 5min homogeneous coloration. •	Add 350 ul of N3 and mix immediately by inverting 34-6 times. Should be cloudy. •	Centrifuge for 10 min (@13000 rpm @RT is fine) •	Apply onto QIAprep spin column. •	Add .75ml of Buffer PE  (next time try adding .5ml PB and centrifuging for 30-60s before this.) Centrifuge for 30-60s •	Discard flow through and centrifuge for 1 min •	Place the column into clean 1.5ml tube and elute with 50ml of buffer EB. Let sit for 1min. Centrifuge for 1 min. o	REMOVE CAP o	PUT EB AT CENTER of column.