"
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.
20/07
Verification of the clonage.
- Week 7 (22-28 July)
22/07
Assembling PhiC31*...
August 2013
- Week 8 (29-4 August)
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
- 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 Tp901.1 cloning
- correct inserts for Bxb1 after E/P restriction
20/08
Cloning :
-Bxb1 with terminator
-RBS+FimE+terminator with weak promoter
-weak promoter with RBS
21/08
Verification of Tp901.1 and PhiC31* in Strataclones by restriction with ClaI, NcOI and HindIII. Results are good.
22/08
In vivo amplification of PhiC31* and Tp901.1 in Strataclones
23/08
Gel extraction of PhiC31* and Tp901.1 from Strataclones
12/08
New PCR to extract mutant PhiC31
14/08
Gel extraction of mutant PhiC31 and transfer into strata clone.
15/08
Cloning:
- Tp901.1 in pSB1C3
- Bxb1 in Psb1C3
