Team:Exeter/lab book/gibs/wk8

Operon Construction  |  Glycobase

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EcoRI and PstI digest of genes (wbnJ, wbnK, wbbC(d) & wfcA)- for checking on gel

Digestion protocol

Water to 20 µl

3A assembled genes to upstream RBS (BBa_B0034)

BBa_B0034 + wbnJ in pSB1K3

BBa_B0034 + wbnK in pSB1K3

BBa_B0034 + wfcA in pSB1K3

BBa_B0034 + wbbC(1) in pSB1K3

BBa_B0034 + wbbC(2) in pSB1K3

BBa_B0034 + wbbC(3) in pSB1K3

3A assembly

Digest concentrations and volumes were altered like so:

DNA – 500 ng

Enzyme A – 0.5 µl

Enzyme B – 0.5 µl

NEBuffer 2 – 2 µl

BSA – 0.2 µl

Water – to 20 µl

Ran gel of PCR product from 24.8.12 (not successful PCR)

PCR using BL21 genomic DNA (extracted previously by Alex B. and Liam)

Control - 0.3 µl DNA, 34.7 µl water

Genomic – 5 µl DNA, 31 µl water

3 step PCR

2 step PCR

PCR setup

98°C for 30s

98°C for 10s, 50°C for 30s, 72°C for 20s – X 5

98°C for 10s, 72°C for 30s – X 25

72°C for 10mins

Hold temp. 4 °C

Ran PCR gel – success! BL21 genomic DNA was the key.

Transformed extra vectors from plates – standard procedure

BioBrick extraction

Transformation of competent cells

PCR purification of wbbC PCR product

Step 1 225 µl buffer PB added to 45 µl product

Additional step after 5 750 µl PE added. Centrifuged 1 min, 13000 rpm, rt. Discarded flow through

Step 7 30 µl water added. Stood for 1 minute. 20 µl extra water added (water was not on centre of membrane)

Stored at -20 °C

PCR for Gibson assembly

Fragments were primed with overlap primers for making three operon constructs

Fragments and volumes (to get 50 ng/µl):

Pbad (large) - 1 µl

wbnJ – 0.15 µl

wbbC - 1 µl

wfcA for operons 1 and 3 – 0.15 µl

wbnK for 2 and 3 – 0.19 µl

Double terminator for 1, 2 and 3 (2 and 3 will be the same, it joins to wbnK in both) – 1 µl

(N.B. dilutions of DNA were made to reach appropriate concentration – these dilutions were used for all following PCR reactions which is why each following PCR write-up states 1 µl of DNA was used)

water to 50 µl

3 step PCR

2 step PCR

PCR setup:

98°C for 30s

98 °C for 10s, 50°C for 30s, 72°C for 20s – X 5

98°C for 10s, 72°C for 30s – X 25

72°C for 10mins

Hold temp. 4 °C

**Thursday 30.8.12**

Miniprep of vectors according to Fermentas protocol

Step 745 µl water added, followed by another 5 µl

Gibson PCR

Pbad L, wbbC, wfcA 1 & 2, wbnK 1

DNA 1 µl, water 33 µl

3 step PCR

PCR setup:

98°C for 30s

98 °C for 10s, 50°C for 30s, 72°C for 20s – X 5

98 °C for 10s, 70°C for 30s, 72°C for 20s – X 5

98 °C for 10s, 72°C for 30s – X 20

72 °C for 10mins

Hold temp. 4 °C

Ran gel – no luck