Team:Exeter/lab book/gibs/wk2

Operon Construction  |  Glycobase

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Protocol as on 11.7.12 (basic transformation procedure.)

TetrRBS and RBS bio brick sequences resuspended in 10µl water

Resuspended DNA (1µl) added to top10 competent cells (25µl)

Incubated on ice for 30 minutes

42 °C heat shock for 30 seconds

Incubated on ice for 2 minutes

S.O.C. Medium aseptically added (250µl)

Incubated in shaking incubator - 37 C, 220 rpm, 1 hour

LB(amp) (1:1000) plates made

Spread plates of 20µl and 100µl transformed E. coli made.

Overnight incubation at 37 °C

17.7.12

Made up LB-agar for later use

Chose 3 colonies from each 100 µl spread plate.

Colonies transferred by pipette tip into 5 ml LB(amp) broth (1:1000 ratio)

Incubated at 37 °C, 220 rpm, overnight

18.7.12 (miniprep attempt)

Centrifuged tubes, 4℃, 10 minutes, 3901 rcf

Pellet was considered too small, probably due to shaking incubator turning off

4 colonies were selected from plates, put in broth and then incubated (as in 17.7.12)

19.7.12 (miniprepping)

Transferred cultures to identical falcon tubes to remove pipette tips

Centrifuged at 3901 rcf for 5 minutes, 4 °C

Supernatant tipped away

250µl resuspension solution added

Pellet resuspended by gentle pipetting up and down

250 µl lysis solution added

Tubes inverted

350 µl neutralisation solution rapidly added

Tubes inverted

Centrifuged at 13000rpm, 5 minutes, 4 °C

Supernatant transferred to geneJET miniprep column

Centrifuged at 13000rpm, 1 minute

Flow through discarded

500 µl wash solution added

Centrifuged at 13000rpm, 30 seconds

Discard flow through

500 µl wash solution added

Centrifuged at 13000rpm, 1 minute

Discard flow through

Centrifuged at 13000rpm, 1 minute

Transferred column to a new 1.5 ml eppendorf

50 µl elution buffer added

Incubated for 2 minutes, room temperature

50 µl elution buffer added

Incubated for 2 minutes at room temperature

Centrifuged for 2 minutes, 13000 rpm

20 µl milli-Q water added

Centrifuged for 2 minutes, 13000 rpm

Columns discarded

Eluted DNA put on ice for analysis with nanodrop spectrophotometer and then stored at -20 °C

Plasmids digested with following:

Plasmid DNA - 6.5 µl (500ng from largest volume)