208

Main purposes today

New PCR amplification of USER fragments

Procedure

PCR

MasterMix x 17 was made to each tube 3ul of Fw and 3 ul of rv primer was added as well as 1ul DNA template

every fragment was run 2 min 98 C followed by 35 cycles of

10 sec 98 C

1 sec 65 C ramp by 0.1C/s down to

10 sec of 55C

3 min 72C

after repetitions, 5 min of 72 C

prepared

• 1l of 10x TBE
• 200ml of 2% agarose solution and added 10 ul EtBr
• casting 3x 2% gels and 1x1% gel
• 200ml EDTA stock solution 0.5 M from powder, dissolved at pH 8 and later equilibrated to pH 7
• 200ml LB medium w. agar and kanamycin
• autoclaved 1.5l of LB medium
• new batch of competent cells
• glycerol stock from I14032

run gel of todays PCR

got SFGFP for TAT and SFGFP for Sec

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