"

From 2012.igem.org

Weeks:

June			July				August				September				October				November

Week 12: 27th August - 2nd September

Monday 27th

Preparation of different cloning:

• Amplification of IAAH and IAAM with new primers
• Amplification of citrine, sfGFP and mCFP with kozac sequence
• Digestion of GFP-AID, BOO34 GB, E1010 GB, R0010 GB, B0015 GB, GB insert in EcoRI PstI

New cloning attempt of IAAH and IAAM in pSC2+

Tuesday 28th

• Preparation of digested pSB1C3 in high quantity. Digestion of pSC2+
• Digestion of the IAAH-IAAM, citrine, sfGFP, mCFP

Ligation of all the parts digested the day before and the day. Transformation using the new electrocompetent cell protocol. Concentration before plating and incubation overnight.

Ligation of TirI using the in-fusion protocol and electroporation

New transformation of pSB1C3 from plates

New culture of pSC2+ BB for midi-prep the day after

Wednesday 29th

All the plates contained cloned. The color of the GB inset indicates that the insertion might have been efficient for most of the clonings.

Colony PCR of 8 clones of each constructs carried out.

GFP-AID	pSC2+	clones XXXXX
citrine	pSC2+	clones XXXXX

<td><tr>GG insert</tr><tr>pSC2+</tr><tr>clones XXXXX</tr></td> <td><tr>sfGFP</tr><tr>pSC2+</tr><tr>clones XXXXX</tr></td> <td><tr>mCFP</tr><tr>pSC2+</tr><tr>clones XXXXX</tr></td> <td><tr>GG insert</tr><tr>pSB1C3</tr><tr>clones XXXXX</tr></td> <td><tr>E1010</tr><tr>pSB1C3</tr><tr>clones XXXXX</tr></td> <td><tr>B0034</tr><tr>pSB1C3</tr><tr>clones XXXXX</tr></td> <td><tr>B0015</tr><tr>pSB1C3</tr><tr>clones XXXXX</tr></td> <td><tr>R1010</tr><tr>pSB1C3</tr><tr>clones XXXXX</tr></td> <td><tr>GFP-AID</tr><tr>pSB1C3</tr><tr>clones XXXXX</tr></td> <td><tr>TirI</tr><tr>pSB1C3</tr><tr>clones XXXXX</tr></td>