"
Team:INSA Toulouse/contenu/lab practice/notebook/calendar/recomb
From 2013.igem.org
Calendar
Recombinases
July 2013
- Week 6 (15-21 July)
15/07
Assembling Bxb1 with terminator by 3A assembling into pSB1A3
Assembling FimE recombinase with rbs by 3A assembling into pSB1C3
Mutagenese of PhiC31 from the Suiti Parts (A24) to delete the EcoRI restriction site
PCR to extract Tp901.1 from the Bonnet parts
16/07
Purification of the PCR and verification by gel.
Miniprep for the verification of the clonage, Bxb1.term and rbs.FimE.
For the mutagenese of PhiC31, restriction by DpnI and transformation.
17/07
Assembling Tp901.1 with terminator by 3A assembling.
PCR of extraction of the vector A24 with Phic31 mutated.
18/07
Assembling PhiC31 mutated with the terminator into pSB1C3.
19/07
Assembling of rbs.FimE with terminator into pSB1C3.
Arrival of the synthesis.
20/07
Verification of the clonage.
- Week 7 (22-28 July)
22/07
Assembling PhiC31*...
30/7
PCR to extract the recombinase PhiC31 (after mutation to delete the EcoR1 site).
31/07
0,8% gels to confirm:
- mutation and extraction of PCR products : some correct inserts
Cloning:
- PhiC31 in PSB1C3 carrying a terminator
- PhiC31 + terminator in PSB1K3 with 3A method
- PhiC31 in PSB1C3
August 2013
- Week 9 (5-11 August)
05/08
Minipreps then 0,8% gel : failed.
08/08
Still trying to clone : PhiC31 in PSB1C3
09/08
Minipreps and 0,8% gel:
- correct length for Phic31, but not the right sequence according to more restrictions.
- Week 10 (12-18 August)
- Week 11 (19-25 August)
19/08
0,8% gel:
- no insert for tp901 cloning
- correct inserts for bxb1 after E/P restriction
12/08
New PCR to extract mutant PhiC31
14/08
Gel extraction of mutant PhiC31 and transfer into strata clone.
15/08
Cloning:
- Tp901 in pSB1C3
- Bxb1 in Psb1C3
