August/5 August 2009

To All From Tadasi I checked these following parts today.

BBa_C0062 BBa_F2620 BBa_F2621 BBa_F2622 BBa_C0171 BBa_C0179 BBa_C0079 BBa_F1610 BBa_C0061 BBa_C0161 BBa_C0070 BBa_C0078 (mainly from "signal senders"and "signal receivers")

5th.August

wet work

1.Mini prep

Culture broth (5ml) was centrifuged (15K,1min, room temperature) to harvest the cells. The cells ware suspended in 250μl of P1　for cell lysis and cutting RNA. 250μl of P2 was added, and tubes ware turned over 4,5 times. Then,350μl of N3 was added,and tubes ware turned over 4,5 times. those suspension ware on ice for 3min. They ware centrifuged (15K,10min,4°C). Those supernatants ware applied to blue columns.Then they ware centrifuged(13K,1min,room temperature) and those solution ware applied and centrifuged (13K,1min,room temperature)again to collect those DNAs. Those resulting pellets ware suspended in 500μl of buffer PB, and they ware centrifuged(13K,1min,room temperature). Those resulting pellets ware suspended in 750μl of PE, and they ware centrifuged(13K,1min,room temperature). Those resulting pekkets ware centrifuged(14K,3min,roomtenperature)again to remove PE. 70μl of buffer EB was applied those columns,and left for 5min.Then,they ware centrifuged(14K,1.5min,room temperature) Those resulting liquids ware tested with Nano Drop.

Result plate2 8-E 78.6ng/μl plate1 8-I 88.0ng/μl plate1 8-K 91.0ng/μl plate1 12-M 89.7ng/μl 2.Transformation plate3 18-0 (BBa_K143032) was transformed.

3.Cutting with restriction enzyme About mole, inserted part is needed from three to six times as much as vector. 20uL (dH2O, enzyme , Vector , Insert) (enzyme : 0.5~1.0 ul) 37°C, 3hr

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