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Team:Evry/Notebook/w9
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| - | <h1> | + | <h1>Week 9: 6th August - 12th August</h1> |
| - | < | + | <h2>Monday, 6th August</h2> |
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| - | < | + | <h3>Reporter test</h3> |
| + | 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. | ||
| + | |||
| + | <h3>GFP BB</h3> | ||
| + | Digestion of GFP BB with EcoRI and PstI, gel extraction and DNA purification. | ||
| + | Goal: insertion in pCS2+ BB | ||
| + | Issue: Not enough DNA for ligation. | ||
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| + | Mini-prep pCS2+-dendra2 and pCS2+-mcherry checked by gel.<br> | ||
| + | inolation for midi-prep pNHK60 (Tir1/GFP-aid),<br> | ||
| + | inoculation for mini-prep pSB1C3 K515100 (IaaH+IaaM).<br> | ||
| + | |||
| + | <h2>Tuesday, 7th August</h2> | ||
| + | </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> | ||
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| + | <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 | ||
</html> | </html> | ||
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/ulMini-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 K515100IaaM 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 testMidi-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 pNHK60Elution 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
