Team:Paris Liliane Bettencourt/Notebook/2011/08/11/

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Team IGEM Paris 2011

Contents

Cyrille

After a long analysis, we discovered that we were working with the wrong plasmid map. Then the interpretation of the manipulations become more clear. Here is the new plasmid map:

the expected plasmid map
The original plasmid map
The plasmid map if the wrong site is digested

Then, we reanalyze the results of the manipulation of the 05/08 and discovered that we may had the good results.

The tubes of 5 and 6, that where identified to be the uncorrect sequence this time are supposed to be the good ones. They where recollected from the trash bin.

To check the presence of the expected plasmid in these tube we will proceed to a control digestion. This time, we want to verify the presence of all the interesting restriction sites. Then we will load a gel.

0.5 µg of DNA from the tubes will be digested into: 

a - No digestion 
        -> one band expected at 5.9kb
        -> verify the presence of the plasmid and the pollution of the MP
b - EcoRI (NEB)   
        -> one band expected at 5.9kb (a bit upper than the previous one, because it is linear)
        -> Verify the presence only one EcoRI site in the plasmid
c - EcoRI and EcoRV (NEB)
        -> One band at 839bp and one at 5,1kb (if uncorrect muation 2.2kb and 3.7kb) (if other map with "correct": 2.2kb and 3.7kb and "uncorrect": 5.08kb 839bp)
        -> Verify that the good EcoRI has been eliminated
d - PstI and EcoRV (NEB) 
        -> One band at 5,05kb and one at 878bp (idem for the uncorrect mutation) (if other map with both "correct" and "uncorrect" mutation 2.3kb and 3.7kb)
        -> Verify that the plasmid is assembled the way we think it is
        -> Verify that the PstI site is functionnal

PstI, EcoRI and PstI works in NEBuffer3. The digestion will be done for one hour at 37°C in 10µL

The tube composition is: Tube 6: 203 ng/µL (ratio 2,1) Tube 7: 480 ng/µL (ratio 2,22)

Due to the bad ratio of the second tube, there are probably proteins, and the quantity of DNA is probably underestimated. We will consider it as the fisrt tube.

Then:

a b c d
DNA 2,5µL 2,5µL 2,5µL 2,5µL
NEBuffer3 . 1µL 1µL 1µL
H20 7,5µL 5,5µL 4,5µL 4,5µL
EcoRI . 1µL 1µL .
EcoRV . . 1µL 1µL
PstI . . . 1µL


The digestion has been held 2 hours. Then the results has been loaded on a gel. The gel is not clean but the result for the tube 6 is the result expected.

ladder, 5a; 5b, 5c, 5d, 6a, 6b, 6c, 6d, ladder

The band of the line 6 has been extracted and will be purified.

band 6a extracted

The Gel extraction and the original miniprep sample has been transformed and plated. The results were analyszed the day after.

Kevin

TECAN from PCR Purification DL Plasmids

  • 1=LacI:GFP

1.4 : 176,7 ng/uL - 1.5 : 179,2 ng/uL

  • 2=tetR:YFP

2.4 : 181,5 ng/uL - 1.5 : 138,4 ng/uL

  • 3=LacO array

3.7 : 109,8 ng/uL - 3.8 : 105,8 ng/uL

  • 4=tetO array

4.7 : 85 ng/uL - 4.8 : 92 ng/uL

Digestion

Digestion of plasmids with Fast Digest XS restriction enzyme.
0,2ng PCR Product

Gel verification of digestion

Gel of digestion - Column 1.4 - 1.5 - 2.4 - 2.5 - 3.7 - 3.8 - 4.7 - 4.8 / S27 digested XS
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