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From 2010.igem.org

Contents 1 Johan 1.1 Visualized coommasie gel 1.2 Site-directed mutagenesis 2 Andreas 2.1 Clonings 2.1.1 Transformations 2.1.2 Colony verification 2.1.2.1 Colony PCR 2.1.2.2 Restreakings 2.1.3 ON cultures

Johan

Visualized coommasie gel

(7 July)

Lane 1: buffer, 2-5: Nina's IgG Protease (0h, 1h, 2h, 3h after IPTG induction), 6-9: my bFGF (0h, 1h, 2h, 3h after IPTG induction), 10: ladder.

bFGF is fused to GST in the vector that was used, with a total size of ~40 kDa, thus showing the correct size on the gel.

Site-directed mutagenesis

• Performed site-directed mutagenesis on bFGF from received vector, and left with Dpn I overnight. (protocol)

• Forward primer

Mol weight (g/mol): 14784,77

Concentration (pmol/µl): 163,08

163E-12 mol/µl * 14789 g/mol = 2,4 µg/µl

To get 125 ng/µl: 2,4E-6/125E-9 = 19

A small volume was diluted 19x and 1 µl was used in the PCR

• Reverse primer

Mol weight (g/mol): 14743,71

Concentration (pmol/µl): 119,37

119E-12 mol/µl * 14743 g/mol = 1,75 µg/µl

To get 125 ng/µl: 1,76E-6/125E-9 = 14

A small volume was diluted 14x and 1 µl was used in the PCR

• dsDNA Template

Concentration: ~300 ng/µl.

A small volume was diluted 30x and 1 µl was used in the PCR

Andreas

Clonings

Transformations

Continued from 12/7.

Growth on all plates. White and red colonies on all pSB1x3 plates, with red colonies being negative clones re-ligated with [http://partsregistry.org/wiki/index.php?title=Part:BBa_J04450 BBa_J04450] RFP cassettes, and white colonies being our positive clones carrying insert of interest.

Colony verification

Two positive clones were picked from each construct and dissolved in 10 μl LB.

Colony PCR

Colony PCR was performed on all pEX-carried inserts. No colony PCR was performed on pSB1x3 clones as the verification primers were not available at the time.

PCR tubes - illustra PuReTaq Ready-To-Go™ PCR Beads:

• 1 μl 10 μM forward primer (pEXf)
• 1 μl 10 μM reverse primer (pEXr)
• 0.5 μl LB cell suspension
• 22.5 μl dH₂O

PCR settings

1. 95°C - 5:00
2. 95°C - 1:00
3. 60°C - 0:30
4. 72°C - 1:30 (Cycle to step 2. 29 times)
5. 72°C - 10:00
6. 15°C - ∞

PCR samples stored in -20°C for later gel separation.

Restreakings

Picked pSB1x3 clones were restreaked onto 1/4 or 1/2 LB agar plates with appropriate antibiotics to verify that the picked clones were indeed positive (RFP fluorescence is sometimes difficult to identify). Plates incubated ON in 37°C.

ON cultures

Although not yet colony-PCR verified, ON cultures were set of the following constructs to save time:

• BL21 - pEX.BBa_J18930-32 in 3 ml LB with 100 μg/ml Amp and 1 % glucose to prevent protein expression leakage.
  • 37°C, 250 rpm, ON
• Top10 - pEX.BBa_J18930-32, .SOD & .yCCS grown in 5 ml LB + 100 μg/ml Amp.
  • 37°C, 250 rpm, ON