Difference between revisions of "Team:Pasteur Paris/Week 13"

Line 107: Line 107:
 
  <li>Treatment of the yeast using a homemade Zymolyase solution&nbsp;:</li>
 
  <li>Treatment of the yeast using a homemade Zymolyase solution&nbsp;:</li>
 
  <ol>
 
  <ol>
−
   <li>pH7,5 PBS 5mL</li>
+
   <li> PBS pH 7.5 5 mL</li>
 
   <li>glucose 5% to make a 2%  solution</li>
 
   <li>glucose 5% to make a 2%  solution</li>
−
   <li>Zymolyase 0.1g (0.012-0.022mg/mL)</li>
+
   <li>Zymolyase 0.1 g (0.012-0.022 mg/mL)</li>
−
   <li>Distilled water free RNAs/DNAs 1mL</li>
+
   <li>Distilled water free RNAs/DNAs 1 mL</li>
 
   <li>MidiPrep of pRS415</li>
 
   <li>MidiPrep of pRS415</li>
 
  </ol>
 
  </ol>

Revision as of 03:05, 19 September 2015

Week 1

Week 2

Week 3

Week 4

Week 5

Week 6

Week 7

Week 8

Week 9

Week 10

Week 11

Week 12

Week 13

Week 14

Week 15

Week 13

08/24 - 08/28


Yeast Assembly

  • Treatment of the yeast using a homemade Zymolyase solution :
    1. PBS pH 7.5 5 mL
    2. glucose 5% to make a 2% solution
    3. Zymolyase 0.1 g (0.012-0.022 mg/mL)
    4. Distilled water free RNAs/DNAs 1 mL
    5. 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 7500 bp which 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    A second trial has been done and TE 8.1 has been replaced by DNAse RNAse free water.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 :
      1. TPA (0.1 g) + Na2H2PO4(0.5 g) in 150 ml of water: complete TPA solubilization.
      2. TPA (0.1 g) + NaOH(0.5 g) in 150 ml of water: complete TPA solubilization.
  • Fluorescence measurements :
    1. no TPAOH was observed on our measurements, the reaction worked.


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