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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.
August 2013
- Week 8 (29 July - 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 on PhiC31+terminator in pSB1K3 and PhiC31 in pSB1C3 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 & minipreps of PhiC31* and Tp901.1 in Strataclones
23/08
Gel extraction of PhiC31* and Tp901.1 from Strataclones
24/08
RBS+FimE+terminator confirmed by sequencing.
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
September 2013
- Week 12 (25 August-2 September)
26/08
Cloning
-Purified PhiC31* in pSB1C3
-Purified Tp901.1 in pSB1C3
27/08
Minipreps and gel verification on PhiC31* and Tp901.1 with EcoRI/PstI: failed for the most part.
28/08
Further verifications on Tp901.1 and PhiC31* with HindIII, NcoI, EcoRI and PstI: failed.
29/08
New gel extractions for PhiC31* and Tp901.1 from Strataclones.
Cloning:
- rbs-FimE-term in Pweak
- Bxb1 in Pweak-rbs
- Rbs-Bxb1 in Pweak
Rbs-Bxb1 confirmed by sequencing, but the terminator is absent.
30/08
Cloning of PhiC31* and Tp901.1 in pSB1C3.
No clone for Pweak- rbs-Bxb1.
Minipreps and gel verification on FimE and Bxb1 with EcoRI/PstI: success.
31/08
No clones for Tp901.1 and PhiC31*. 02/09
Gel extractions and cloning of PhiC31* and Tp901.1 in pSB1K3.
For Pweak-rbs-Bxb1, further verification by restriction with:
- EcoRI
- AvrII/PstI, AvrII cutting in Pweak
Length of the fragment obtained corresponds to what is expected. Success confirmed by restriction.
- Week 13 (3-9 September)
03/09
For PhiC31* and Tp901.1 in pSB1C3, minipreps and digestion by EcoRI and PstI. Results not satisfactory.
For Pweak-rbs-FimE-term, further verification by restriction with:
- EcoRI
- AvrII/StuI, AvrII cutting inside Pweak and StuI inside FimE
Length of the fragment obtained corresponds to what is expected. Success confirmed by restriction.
Rbs-FimE-Term confirmed by sequencing.
04/09
Design of In-Fusion primers for direct assembling of Weak promoter - RBS - Recombinase -Terminator in pSB1C3, concerning PhiC31* and Tp901.1.
Cloning:
-Pweak-rbs-Bxb1 in Terminator
-Pweak-rbs-Bxb1 in psB1C3 and psB1K3
05/09
New gel extractions and cloning of PhiC31* and Tp901.1 in pSB1A3. PhiC31* and Tp901.1 sequences are confirmed in Strataclones by sequenciing.
Minipreps and gel verification on Bxb1 with EcoRI/PstI: Profiles seems weird. Need further verifications.
Pweak-rbs-Bxb1 confirmed by sequencing.
06/09
For PhiC31* and Tp901.1 in pSB1A3, minipreps digested with EcoRI/PstI seems to be ok only for PhiC31*.
For Bxb1, further verification by restriction with:
- EcoRI
- AvrII/ BamHI: BamHI cutting inside Bxb1
The results show that the cloning failed.
08/09
New cloning of Tp901.1 in pSB1C3, pSB1K3 and pSB1A3.
Further verifications with NcoI invalidate previous result on PhiC31* in pSB1A3.
09/09
Pweak-rbs-FimE-Term confirmed by sequencing.
- Week 14 (10-16 September)
10/09
Minipreps on Tp901.1 are wrong.
11/09
Infusion primers received:
-PCR on the differents parts to join: Pweak+rbs - Tp901.1 or PhiC31.1 - Terminator - pSB1C3.
-Infusion reaction for both the recombinases.
-Transformation in Stellar competent cells.
13/09
Minipreps of infusion transformations. Infusion assembly failed.
14/09
New infusion reactions on PhiC31* and Tp901.1 with different concentrations of each part.
15/09
Screening of infusion results by PCR on colonies.
16/09
Migration of PCR screening. Tp901.1 seems to be good.
New infusion reaction for PhiC31*.
- Week 15 (17-23 September)
17/09
Further verifications on Tp901.1 showed wrong results. No good news at all.
New infusion reaction for Tp901.1 with both PCR amplified and linearized plasmids.
PCR sreening on previous PhiC31* infusion reaction.
18/09
PCR screening failed for unknown reasons. Minipreps done on the previous PhiC31* infusion reaction and gave bad results.
- Week 15 (24-30 September)
24/09
New infusion reaction with new primers: Vector+Pweak+RBS - Recombinase - Terminator.
26/09
Minipreps on previous infusion reaction gave bad results. We decide to keep on using original sequences of Tp901.1 and PhiC31 integrases for characterizations.
