Team:Exeter/lab book/gibs/wk4

Operon Construction  |  Glycobase

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4 pm

Inoculated LB(kan) broth with transformed pbad large (BBa_I0500) colonies

**Tuesday 31.7.12**

9 am

Miniprep of pbad large (BBa_I0500)

Protocol as on 27.7.12

Step 10 changed to 9 minutes

Step 22 changed to 20 µl

Step 24 changed to 10 µl

3A assembly for Pbad large-RBS, as on 25.7.12

Digestion:

Upstream

Pbad large DNA (500 ng):

8.68 µl

Enzymes:

as before

Water:

18 µl

Downstream

RBS DNA (500 ng):

2.94 µl

Enzymes:

as before

Water:

14.56

Destination plasmid (kanamycin)

Master mix:

EcoRI-HF:

0.5 µl

PstI:

0.5 µl

10X NEBuffer 2:

5 µl

100X BSA:

0.5 µl

Water:

18 µl

4 µl of master mix added to 4 µl of linear pSB1K3 DNA (100 ng)

Digested at 37 °C for 1 hour

Heat inactivated at 80 °C for 20 minutes

Ligation

Upstream digest:

2 µl

Downstream digest:

2 µl

Plasmid digest:

2 µl

T4 DNA ligase buffer:

1 µl

T4 DNA ligase:

0.5 µl

Water:

2.5 µl

Incubated at room temperature for 1.5 hours

Heat inactivated at 80 °C for 20 minutes

2pm

Sent DNA off for sequencing – Pbad large (and also PlacI-RBS construct)

DNA prepared as per bioline instructions.

3 pm

Transformed as on 25.7.12

Step 7 plated volumes were 50 µl and 150 µl on LB(kan)

**Thursday 2.8.12**

9 am

Miniprep of pBad large-RBS

Protocol as on 17.7.12

Step 22 45 µl water, stand at room temp. For 2 mins

Step 23 centrifuge 13000 rpm, room temperature, 2 minutes

Step 24 5 µl water

2.30 pm

Gel of pbad large-RBS

Digest for gel (using only one enzyme – nanodrop data was uncertain)

DNA (assumed ~100ng/µl):