Team:Paris/August 20

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(Difference between revisions)
(→Screening of the cloning of E0240 and FlhDC+promotor)
(→Screening of the cloning of E0240 and FlhDC+promotor)
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*Dilution 10 and dilution 100
*Dilution 10 and dilution 100
*Spreading of 100 µL of each dilution on a LB plate containing the right antibiotic
*Spreading of 100 µL of each dilution on a LB plate containing the right antibiotic
-
*Overnight incubation
+
*Overnight incubation (37°C)
=Construction of pFlgA - YFP tripart=
=Construction of pFlgA - YFP tripart=

Revision as of 17:28, 20 August 2008

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Contents

Screening of the cloning of E0240 and FlhDC+promotor

Spreading the clones in order to obtain single colonies

Strain Resistance Ligation DNA cloned vector expected size of the fragment amplified by VF & VR mesured size
S159.1 kanamycine L139.1 E0240 (GFP tripart) pSB3K3 1192 bp 1,5 kb
1,1 kb
0,6 kb
S161.1 ampicilline L142.7 FlhDC+promotor pSB1A2 1403 bp 1,4
0,4 kb
0,3 kb

The plates obtained from the speading of yesterday can't be used because there are not single colonies.
We have to try again, but with a stronger dilution of the bacteria or with a smaller volume of spreading.

  • Resuspension of some bacteria from the glycerol stock into 1 mL of LB+antibiotic
  • Dilution 10 and dilution 100
  • Spreading of 100 µL of each dilution on a LB plate containing the right antibiotic
  • Overnight incubation (37°C)

Construction of pFlgA - YFP tripart

Aim : Construction of "pFlgA-RBS-YFP-dbl ter" (pFlgA-E0430/E0432) Part icon regulatory.pngPart icon rbs.pngIcon coding.pngPart icon terminator.pngPart icon terminator.png

Digestion

Measurement of concentration of minipreps

standard protocol

Miniprep Biobrick C° (µg/mL) ratio 260/280
MP164.1 E0422 95 1.69
MP164.2 E422 90 1.76
MP165.1 E0430 131 1.74
MP165.2 E0430 nd nd
MP166.1 E0432 112 1.65
MP166.2 E0432 79 1.6
MP167.1 E0420 199 1.72
MP167.2 E0420 194 1.73
MP168.1 I732078 111 1.65
MP168.2 I732078 104 1.67
MP122.1 E0840 56 1.58
MP122.2 E0840 98 1.63


Digestion

Protocol Digestion

Name Template DNA Description Vol MP (µl) Vol H2O (µl) Enzymes
D166 MP165.1 - FV 7.63 17 EcoRI and XbaI
D167 MP166.1 - BV 8.9 15.8 EcoRI and XbaI
D131 MP122.2 - BV 10.2 14.5 XbaI and PstI


Gel Extraction

File:KR000.jpg
Gel Extraction of D166-D167-D131

Protocol


Well Sample Expected size Measured size
1 1kb ladder
2 MP165.1 2 957 pb
3 MP166.1 2 996 pb
4 MP122.2 2 957 pb
5 no sample
6 D166 885 pb
7 no sample
8 D167 914 pb
9 no sample
10 D131 900 pb
11 no sample
12 100pb ladder
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