Team:Exeter/lab book/gibs/wk4

Operon Construction  |  Glycobase

-

-

-

-

-

-

-

-

-

-

4 pm

Inoculated LB(kan) broth with transformed pBAD large (BBa_I0500) colonies according to protocol

**Tuesday 31.7.12**

9 am

Centrifuged broth tubes for 5 minutes, 3901 rcf, 4 °C

Supernatant discarded

Miniprep of pBAD (BBa_I0500)

Step 4 changed to 9 minutes

Step 1020µl milli-Q water added in place of elution buffer, followed by another 10 µl after incubation and centrifugation

3A BioBrick Assembly for pBAD-RBS (BBa_I0500 + BBa_B0034)

Digestion:

Upstream

pBAD (BBa_I0500) plasmid DNA: 8.68 µl

MilliQ water: 18 µl

Downstream

RBS (BBa_B0034) plasmid DNA: 2.94 µl

MilliQ water: 14.56

Destination plasmid (pSB1K3 – kanamycin resistance) digested according to protocol for linearised backbone

Digested at 37 °C for 1 hour

Heat inactivated at 80 °C for 20 minutes

Ligation according to same protocol

Downstream digest: 2 µl

Plasmid digest: 2 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 with VF2 and VR primers – pBAD (BBa_I0500) according to Bioline instructions

3 pm

pBAD-RBS construct transformed

2 µl of construct DNA added to competent cells

Plated volumes were 50 µl and 150 µl on LB(kan)

**Thursday 2.8.12**

9 am

Broth tubes centrifuged for 5 minutes, 3901 rcf, 4 °C

Supernatant discarded

Miniprep of pBAD-RBS (BBa_I0500 + BBa_B0034)

Step 1045µl milli-Q water added in place of elution buffer, followed by another 5 µl after incubation and centrifugation

2.30 pm

Gel of pPAD large-RBS (BBa_I0500 + BBa_B0034)

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

DNA (assumed ~100ng/µl): 5 µl

EcoRI-HF: 1µl

100X BSA: 0.5 µl

10X NEBuffer 2: 5 µl

Water: 11 µl

Incubated at 37 °C for 20 minutes

Run on 1% agarose gel

LB(amp) streak plates made for pBAD-RBS (BBa_I0500 + BBa_B0034) dregs from miniprep.