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Week 9: 6th August - 12th August

Monday, 6th August

Reporter test

We had a lot of reporters BB but we didn't know if they're all working. We test them with a electrophoresis: Two RFP, a GFP, a CFP and a YFP worked.


Digestion of GFP BB with EcoRI and PstI, gel extraction and DNA purification. Goal: insertion in pCS2+ BB Issue: Not enough DNA for ligation. Mini-prep pCS2+-dendra2 and pCS2+-mcherry checked by gel.
inolation for midi-prep pNHK60 (Tir1/GFP-aid),
inoculation for mini-prep pSB1C3 K515100 (IaaH+IaaM).

Tuesday, 7th August

Midi-prep pNHK60 (Tir1/GFP-aid): elution in 1ml, 35,2ng/ul
Mini-prep pSB1C3 K515100 (IaaH+IaaM).
Digestion pSB1C3 K515100 (IaaH+IaaM)with EcoRI and PstI then transformation in pCS2+ (amplified with PCR, digested with EcoRI and PstI then purified).

Wednesday, 8th August

Speed vac of pNHK60 (midi-prep 7th August): resuspension in 50ul : 1254,9ng/ul, 260/280nm = 1,79 (tube A9)
inolation for midi-prep pSB1C3 (IaaH+IaaM),

Iaa BB

IaaH PCR: P35 + P33 + pSBIC3 IaaH IaaM K515100
IaaM PCR: P34 + P22 + pSBIC3 IaaH IaaM K515100
IRES PCR: P31 + P32 + pNHK60
Results: Primers are wrong for IaaH and IaaM PCR, IRES PCR is good.

Mutation of TirI

We make two PCR to mutate two nucleotide in TirI sequence:
PCR 1: P1 + P4 + pNHK60
PCR 2: P2 + P36 + pNHK60

Thursday, 9th August

pSB1C3 (IaaH+IaaM)

Recovery of the supernatant for Salkowski test
Midi-prep pSB1C3

Ligation of pCS2+ and Iaa

Digestion of pCS2+ and pCSBAC3 IaaH+IaaM K515100 with EcoRI and PstI.
Purification and ligation.
Gradient PCR for IaaM with primer p35 and p22. and Tir1 with primer p1 and p4

Friday, 10th August


Phenol/chloro gDNA extration of the midiprep of pNHK60
Elution in 20ul TE 1X
Concentration: 2211,9 ng/ul rapports: 260/280=1,62, 260/230=1,86
After Colony PCR: transformation pCS2+/K515100 did not worked.
Gel after the gradient PCR of thursday: extraction of Iaam at 1, 650 bp and Tir1 at 1 kb