Latest revision as of 08:33, 13 August 2012

Weeks:

June			July				August				September				October				November

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.

GFP BB

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

pNHK60 (TIR-GFP-AID)

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

@@ Line 21: / Line 21: @@
 <h2>Tuesday, 7th August</h2>
 </br>
-Midi-prep pNHK60 (Tir1/GFP-aid),<br>
+Midi-prep pNHK60 (Tir1/GFP-aid): elution in 1ml, 35,2ng/ul<br>
 Mini-prep pSB1C3 K515100 (IaaH+IaaM).<br>
 Digestion pSB1C3 K515100 (IaaH+IaaM)with EcoRI and PstI then transformation in pCS2+ (amplified with PCR, digested with EcoRI and PstI then purified).<br>
@@ Line 27: / Line 27: @@
 <h2>Wednesday, 8th August</h2>
+Speed vac of pNHK60 (midi-prep 7th August): resuspension in 50ul : 1254,9ng/ul, 260/280nm = 1,79 (tube A9)<br>
+inolation for midi-prep pSB1C3 (IaaH+IaaM),<br>
 <h3>Iaa BB</h3>
 IaaH PCR: P35 + P33 + pSBIC3 IaaH IaaM K515100 <br>
 IaaM PCR: P34 + P22 + pSBIC3 IaaH IaaM K515100<br>
 IRES PCR: P31 + P32 + pNHK60<br>
+Results: Primers are wrong for IaaH and IaaM PCR, IRES PCR is good. <br>
+<h3>Mutation of TirI</h3>
+We make two PCR to mutate two nucleotide in TirI sequence:<br>
+PCR 1: P1 + P4 + pNHK60<br>
+PCR 2: P2 + P36 + pNHK60<br>
+<h2>Thursday, 9th August</h2>
+<h3>pSB1C3 (IaaH+IaaM)</h3>
+Recovery of the supernatant for Salkowski test<br>
+Midi-prep pSB1C3<br>
+<h3>Ligation of pCS2+ and Iaa</h3>
+Digestion of pCS2+ and pCSBAC3 IaaH+IaaM K515100 with EcoRI and PstI.<br>
+Purification and ligation.<br>
+Gradient PCR for IaaM with primer p35 and p22. and Tir1 with primer p1 and p4<br>
+<h2>Friday, 10th August</h2>
+<h3>pNHK60 (TIR-GFP-AID)</h3>
+Phenol/chloro gDNA extration of the midiprep of pNHK60<br>
+Elution in 20ul TE 1X<br>
+Concentration: 2211,9 ng/ul rapports: 260/280=1,62, 260/230=1,86<br>
+After Colony PCR: transformation pCS2+/K515100 did not worked.<br>
+Gel after the gradient PCR of thursday: extraction of Iaam at 1, 650 bp and Tir1 at  1 kb
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Team:Evry/Notebook/w9

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