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Week 13
08/24 - 08/28
Yeast Assembly
- Treatment of the yeast using a homemade Zymolyase solution :
- pH7,5 PBS 5mL
- glucose 5% to make a 2% solution
- Zymolyase 0,1g (0,012-0,022mg/mL)
- Distilled water free RNAs/DNAs 1mL
- MidiPrep of pRS415
Dilution 1/50 |
DNA Concentration (ng/µl) |
OD(230nm) |
OD(260nm) |
OD(280nm) |
OD(260nm)/OD(230nm) |
OD(260nm)/OD(280nm) |
pRS415 |
6,2 |
0,122 |
0,067 |
0,076 |
1,60 |
1,83 |
MidiPrep has been digested to verified if the DNA correspond to the plasmid of interest or to an other plasmid that is naturally present in yeast.
- BamHI digestion of pRS415 (with the phosphatase step).
We can see the presence of a streak about 7500bp wich seems to correspond to our plasmid pRS415.
- DNA concentration measurement.
|
DNA Concentration (ng/µl) |
OD(260nm) |
OD(280nm) |
OD(260nm)/OD(280nm) |
BBa_J61047 |
0,4 |
0,008 |
0,002 |
3,76 |
- PCR amplification of pRS415 with a mutagenesis kit using TE 8.1.
- PCR amplification of pRS415 with a mutagenesis kit to insert nicks without TE 8.1.
A second trial has been done and TE 8.1 has been replaced by DNAse RNAse free water.
- Gel migration on a 0.7% agarose gel.
- PCR amplification of pRS415 with new recommandations (protocol n°3)
- Gel migration on a 0.7% agarose gel.
- Preparation of electro-competent yeast cells.
- Electroporation of pRS415 and Cre with overlapping sequences.
TPA solubility :
- improvement of TPA solubility in different conditions :
- TPA (0.1g) + Na2H2PO4(0.5g) in 150ml of water: complete TPA solubilization.
- TPA (0.1g) + NaOH(0.5g) in 150ml of water: complete TPA solubilization.
- Fluorescence measurements :
- no TPAOH was observed on our measurements, the reaction worked.
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