Team:Exeter/lab book/gibs/wk9

Operon Construction  |  Glycobase

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Gibson PCR again

Fragments and concentrations as 30.8.12

3 step PCR

Two step PCR

PCR setup:

98 °C for 30s

98 °C for 10s, 69 °C for 30s, 72 °C for 20s – X 5

98 °C for 10s, 72 °C for 30s – X 25

72 °C for 10minutes

Hold temp. 4 °C

Ran gel – only wfcA 1 & 2 worked

**Tuesday 4.9.12**

Mega-miniprep day: Due to various iGEM commitments, an overwhelming number of minipreps was required. LB broth was inoculated 3.9.12 - Four colonies from each plate from all transformations from 21.08.12 onwards were put in broth and miniprepped (see Freddie’s lab book for 3 gene inducible plasmid for details)

Miniprep according toFermentas protocol

**Wednesday 5.9.12**

Used nanodrop spectrophotometer to find concentrations from minipreps the previous day

Gibson PCR of Pbad, wbnJ, wbbC and wbnK 2.

DNA 1 µl, water 33 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 10 minutes

Hold temp. 4 °C

Gel run – no success

**Thursday 6.9.12**

Gibson PCR using Immomix red (2x). Pbad, wbnJ, wbbC, wbnK 2.

Immomix red 25 µl

DNA 1 µl

Primer 1 – 2 µl

Primer 2 – 2 µl

Immomix red 2x – 25 µl

Water - 20 µl

PCR setup:

95 °C for 10 minutes

95 °C for 10s, 55 °C for 30s, 72 °C for 30s – X 25

Hold temp. 4 °C

**Friday 7.9.12**

Ran gel from previous PCR – wbbC worked (although significant blurring – fewer PCR steps may have helped.)

Gibson PCR of Pbad, wbnJ and wbnK 2 using immomix red again

Volumes as on 6.9.12

PCR setup:

95 °C for 10 minutes

95 °C for 10s, 65 °C for 30s, 72 °C for 30s – X 5

4 °C hold step

Ran gel – no success