Team:Exeter/lab book/gibs/wk1

Operon Construction  |  Glycobase

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Dried plasmid DNA of useful parts (chosen parts were a double terminator (BBa_B0014), pBAD weak promoter (BBa_K206001) and pBAD strong promoter (BBa_K206000)) was resuspended according to BioBrick extraction protocol. Remaining DNA was stored at -20°C

Plasmid DNA was transformed into competent cells

-Tube 1: 50 µl E. coli + 1 µl terminator

-Tube 2: 25 µl E. coli + 1 µl pBAD weak

-Tube 3: 25 µl E. coli + 1 µl pBAD strong

250 µl of S.O.C. medium was added to tube 1

125 µl of S.O.C. medium was added to tubes 2 & 3

Tubes were shaken horizontally at 36.8 C, 220rpm for 1 hour

Spread plates of 20 and 100 µl volumes of each plasmid transformed E. coli were prepared on LB(ampicillin) plates

Extra spread plates of 20 and 100 µl volumes of double terminator plasmid were prepared on LB(kanamycin) to check for dual antibiotic resistance.

**Thursday 12.7.12**

Transferred aseptically into 1:1000 ampicillin:LB broth.

Incubated at 37*C, 220rpm overnight.

**Friday 13.7.12**

Cultures were transferred to identical sterile falcon tubes and centrifuged at 21 C, 3900 rcf, for 2 minutes.

Miniprep of plasmid DNA according toFermentas protocol

Step 10 50µl milli-Q water added in place of elution buffer

Tested DNA concentration with nanodrop spectrophotometer. These concentrations were obtained:

pBAD strong -

1. 130.4

2. 105.3

3. 114.2

pBAD weak -

1. 186.4

2. 211.4

3. 170.6

Plasmids digested with following:

Plasmid DNA - 4.2 µl

EcoR1-HF - 0.5 µl

PstI - 0.5 µl

10x NEBuffer 2 - 2.5 µl

100x BSA - 0.25 µl

H2O - 7.95 µl

Incubated for 10 minutes at 37 °C

DNA run on gel by Becca and Alex to test fragments