Team:Exeter/lab book/gibs/wk2

Operon Construction  |  Glycobase

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TetrRBS and RBS (BBa_B0034) BioBricks resuspended and used for competent cell transformation.

1µl of resuspended DNA added to 25µl top10 competent cells.

LB(amp) (1:1000) plates used for 20µl and 100µl spread plates.

**Tuesday 17.7.12**

Previous days spread plate colonies transferred to liquid medium, 1:1000 LB(amp) broth.

**Wednesday 18.7.12**

Centrifuged tubes, 4℃, 10 minutes, 3901 rcf

Miniprep attempt from protocol

Pellet was considered too small, believed to be due to shaking incubator turning off overnight

4 colonies were transferred to liquid medium as on previous day

**Thursday 19.7.12**

Transferred cultures to identical falcon tubes to remove pipette tips

Centrifuged at 3901 rcf for 5 minutes, 4 °C

Supernatant tipped away

Miniprep attempt 2

Step 10 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 from miniprep digested with EcoR1-HF and PstI according to digestion protocol

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

EcoR1-HF - 1 µl

PstI - 1 µl

10x NEBuffer 2 - 5 µl

100x BSA - 0.5 µl

MQ Water - 36 µl

Incubated for 10 minutes at 37 °C

DNA run on gel to test fragments