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 <check to make sure EtOH added?> (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.