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, TetR promoter, LacI/Ara-1 promoter, pBAD/AraC promoter weak and pBAD/AraC promoter strong) was resuspended in 10 µl milliQ water.

DNA was left to resuspend for 5 minutes before being put in eppendorf tubes.

Tubes were centrifuged briefly (around 5 seconds) to spin down liquid.

Plasmid DNA was gently pipetted into volumes of competent Top10 E. coli cells like so:

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

-Tube 2: 25 µl E. coli + 1 µl TetR

-Tube 3: 25 µl E. coli + 1 µl LacI/Ara-1 promoter

-Tube 4: 25 µl E. coli + 1 µl pBad/AraC weak

-Tube 5: 25 µl E. coli + 1 µl pBad/AraC strong

Tubes were kept on ice for 30 minutes

Cells were heat shocked for 30 seconds at 42 *C

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

125 µl of S.O.C. medium was added to tubes 2-5

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

2 LB-agar-antibiotic stocks were made with these ratios:

​1:1000   ampicillin:LB-agar

​1:1000   kanamycin:LB-agar

10 LB(ampicillin) plates were made

2 LB(kanamycin) plates were made

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.

Plates were inverted and incubated overnight at 37 C.

12.7.12

Chose three colonies from each plate.

Transferred aseptically into 1:1000 ampicillin:LB broth.

Incubated at 37*C, 220rpm overnight.

13.7.12

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

250µl lysis solution added

350µl neutralisation solution added quickly

Tubes inverted ~3 times to mix.

Centrifuged at 16100 rcf for 5 minutes, 21 C

Supernatant added to miniprep column

Centrifuged at 16100 rcf, 1 min

Added 500µl wash solution to column

Centrifuged at 16100 rcf, 1 min. Supernatant discarded.

Centrifuged again at 16100 rcf, 1 min

Column switched to new 1.5ml eppendorf

50µl milli-Q water added

Incubated at room temperature (21 C) for 2 mins

Centrifuged for 2 minutes, 16100 rcf.

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

Pbad strong -

1. 130.4

2. 105.3

3. 114.2

Tetr -

1. 76.8

2. 60.9

3. 78.0

Plac -

1. 280.7

2. 272.2

3. 299.2

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