Team:Paris/July 30

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==Analysis of yesterday DNA digestion==
==Analysis of yesterday DNA digestion==
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Revision as of 17:08, 5 August 2008

← Yesterday

↓ Calendar ↑

Tomorrow →



Contents

Results of transformations

Ligation name Description Antibio Nb colonies Comments
L100 rbs TetR - ECFP
D110 (BV) - D130 (BI) 		Amp 0 After another night nothing was osberved
-> To do again
L101 rbs TetR - GFP tripart
D110 (BV) - D131 (BI) 		Amp 1 After another night nothing was observed
-> To do again
L102 Strong rbs - YFP
D129 (BV) - D118 (BI) 		Amp +/- 100
L103 Strong rbs - GFP
D129 (BV) - D122 (BI) 		Amp a lot
L104 Strong rbs - lasR activator
D129 (BV) - D114 (BI) 		Amp a lot
L105 Strong promoter - ECFP
D123 (BV) - D130 (BI) 		Amp a lot
L106 Strong promoter - gfp Tripart
D123 (BV) - D131 (BI) 		Amp a lot
L107 Strongest promoter - ECFP
D103 (BV) - D131 (BI) 		Amp a lot
L108 n°2
(the right one) 		Strong promoter - gfp Tripart
D103 (BV) - D130 (BI) 		Amp +/- 100
L109 n°1 Strong promoter - ecfp
D124 (BV) - D130 (BI) 		Amp +/- 30 To do again
L109 n°2 Strong promoter - ecfp
D124 (BV) - D130 (BI) 		Amp a lot To do again
L110 Medium promoter - gfp Tripart
D124 (BV) - D131 (BI) 		Amp a lot
L111 Weak promoter - ECFP
D104 (BV) - D130 (BI) 		Amp a lot
L112 Weak promoter - gfp
D104 (BV) - D131 (BI) 		Amp a lot
L113 AracpBAD - ecfp
D126 (BV) - D130 (BI) 		Kan 1 After another night nothing was observed
-> To do again
L114 AracpBAD - gfp tripart
D126 (BV) - D131 (BI) 		Kan 2 After another night nothing was observed
-> To do again
L115 pLas - ECFP
D105 (BV) - D130 (BI) 		Amp a lot
L116 pLas - gfp Tripart
D105 (BV) - D131 (BI) 		Amp a lot
L117 yfp - Double Terminator
D117 (FI) - D125 (FV) 		Amp +/- 30
L118 rfp - Double Terminator
D121 (FI) - D125 (FV) 		Amp +/- 30
L119 lasR activator + LVA - Double Terminator
D113 (FI) - D125 (FV) 		Amp +/- 30
L120 tetR repressible - ECFP
D106 (BV) - D130 (BI) 		Amp a lot
L121 Strong promoter - gfp tripart
D106 (BV) - D130 (BI) 		Amp a lot
L122 RBS-lasI - ecfp
D107 (BV) - D130 (BI) 		Amp 0 After another night nothing was observed
-> To do again
L123 RBS lasI - ecfp
D107 (BV) - D131 (BI) 		Amp 1 After another night nothing was observed
-> To do again
L124 Strongest RBS - rfp
D102 (BV) - D122 (BI) 		Amp a lot
L125 Strongest RBS - yfp
D102 (BV) - D118 (BI) 		Amp a lot
L126 Strongest RBS (1)- LacR activator (+LVA)
D102 (BV) - D118 (BI) 		Amp no dish found!!! To do again
L127 gfp (1)- Double terminator
D119 (FI) - D125 (FV) 		Amp no dish found!!! To do again
Controls
C1

D110

Amp 0 -
C2

D129

Amp 5 -
C3

D123

Amp +/- 100 -
C4

D103

Amp +/- 20 -
C5

D124

Amp +/- 100 -
C6

D104

Amp +/- 100 -
C7

D126

Kana 0 -
C8

D105

Amp +/- 20 -
C9

D125

Amp 0 -
C10

D106

Amp 0 -
C11

D107

Amp 0 -
C12

D102

Amp +/- 10 -
Positive control

puc19

Amp 155 (transformation
efficiency:1.5*10^7/ug) 		-


Analysis of yesterday DNA digestion

The digested DNA of yesterday was analysed one more time by electrophoresis on a 0.8% agarose gel (about 30 minutes at 100 W).

  • The ladder used was the 1 kb DNA ladder (New England Biolabs).
  • 5 µL of each sample with 1 µL of loading dye were loaded.'
KR000082.jpg


Name BioBrick Tube N° Enz 1 Enz 2 Obs Exp Size Mea Size Conc (ng/µl) Band
D101 B0034 1 EcoRI XbaI FV 2076 bp 2000 bp 6 10
D102 B0034 1 SpeI PstI BV 2077 bp 2000 bp 8 11
D105 R0079 1 SpeI PstI BV 2222 bp 2000 bp 6 13
D106 R0040 1 SpeI PstI BV 2119 bp 2000 bp 6 12
D108 S03154 1 XbaI PstI BI 707 bp 0 (failed) 14
D111 S03879 1 XbaI PstI BI 725 bp 600 bp 2 15
D115 C0179 2 EcoRI SpeI FI 746 bp 0 (failed) 4 & 5
D116 C0179 2 XbaI PstI BI 745 bp 700 bp 2 2 & 3
D117 E0030 1 EcoRI SpeI FI 746 bp 600 bp 10 16
D128 B0030 2 EcoRI XbaI FV 2079 bp 2000 bp 8 6 & 7
D129 B0030 2 SpeI PstI BV 2080 bp 2000 bp 8 8 & 9

==> Conclusion :Each of the samples was succesfully digested and purified except for the sample D108. It seems that the QIAprep columms (from the QIAGEN Minipreps kit) can be used instead of the QIAquick columms (for DNA Gel Extraction).

PCR Screening of Ligation Transformants

Use of 8 clones of Ligation transformants for screening PCR


Protocol of screening PCR

  • Mix
Name Vol (µl) Concentration
Quick Load 25µl 2X
OligoF_VF2 (O18) 1µl 10µM
OligoR_VR (O19) 1µl 10µM
water 23µl


  • 50µl of Mix PCR by tube/clone
  • one toothpick of each clone's colony by tube
  • Program : Annealing 55°C - Time élongation 1'30" - Number cycle : 29


Conditions of electrophoresis

  • 10µl of ladder 1 kb
  • 15µl of screening PCR (gel n°1, 2, 3(9-17), 4, 5, 6, 7, 8, 9, 10, 11)
  • 10µl of screening PCR (gel n°3(1), 13, 14)
  • migration ~30min at 100W on 0,8% gel

Results

PCR1_’’’L102(1-8)’’’ PCR2_’’’L103(1-8)’’’ PCR3_’’’L104(1-8)’’’ PCR4_’’’L105(1-8)’’’
Expected size Measured size Band Expected size Measured size Band Expected size Measured size Band Expected size Measured size Band
1045 pb 1000 pb 2-->9 1003 pb 1000 pb 10-->17 1078 pb 1000pb 2-->9 1239 pb 1200 pb 10-->17
Gel 1 : L102-L103
Gel 2 : L104-L105
PCR5_’’’L106(1-8)’’’ PCR6_’’’L107(1-8)’’’ PCR7_’’’L108.1(1-8)’’’ PCR8_’’’L108.2(1-8)’’’
Expected size Measured size Band Expected size Measured size Band Expected size Measured size Band Expected size Measured size Band
1200 pb 1200 pb 2-->9 1239 pb 1200 pb 10-->17 1200 pb 1200 pb 2-->9 1200 pb 1200 pb 10-->17
Gel 3 : L106-L107
Gel 4 : L108.1-L108.2
PCR9_’’’L110(1-8)’’’ PCR10_’’’L111(1-8)’’’ PCR11_’’’L112(1-8)’’’ PCR12_’’’L115(1-7)’’’
Expected size Measured size Band Expected size Measured size Band Expected size Measured size Band Expected size Measured size Band
1200 pb 1100 pb 2-->9 1239 pb 1100 pb 10-->17 1200 pb 1000 pb 2-->10 1239 pb 700 pb 11-->16
Gel 5 : L110-L111
Gel 6 : L112-L115
PCR13_’’’L115(8)’’’ PCR14_’’’L116(1-8)’’’ PCR15_’’’L117(1-6)’’’
Expected size Measured size Band Expected size Measured size Band Expected size Measured size Band
1239 pb 1200 pb 2 1200 pb 1200 pb 3-->10 1046 pb 1000 pb 11-->16
Gel 7 : L115-L116-L117
PCR16_’’’L117(7-8)’’’ PCR17_’’’L118(1-8)’’’ PCR18_’’’L119(1-5)’’’
Expected size Measured size Band Expected size Measured size Band Expected size Measured size Band
1046 pb 1000 pb 2-->3 1004 pb 1100 pb 4-->11 1079 pb 1200 pb 12-->16
Gel 8 : L117-L118-L119
PCR19_’’’L119(6-8)’’’ PCR20_’’’L120(1-8)’’’ PCR21_’’’L121(1-4)’’’
Expected size Measured size Band Expected size Measured size Band Expected size Measured size Band
1079 pb 1100 pb 2-->4 1239 pb 1000 pb 5-->12 1200 pb 1200 pb 13-->16
Gel 9 : L119-L120-L121
PCR22_’’’L121(5-8)’’’ PCR23_’’’L122(1-8)’’’ PCR24_’’’L125(1-4)’’’
Expected size Measured size Band Expected size Measured size Band Expected size Measured size Band
1200 pb 1000 pb 2-->5 1239 pb 1000 pb 6-->12 1045 pb 1000pb 13-->16
Gel 10 : L121-L122-L125
PCR25_’’’L125(5-8)’’’ PCR26_’’’L109.1(1-7)’’’ PCR27_’’’L109.2(1-8)’’’
Expected size Measured size Band Expected size Measured size Band Expected size Measured size Band
1045 pb 1000 pb 2-->5 1239 pb 1100 pb 2-->9 1239 pb 1100 pb 10-->17
Gel 11 : L125
Gel 14 : L109.1-L109.2


==> Conclusion : with the PCR, we have check that the transformant bacteria contain insert. (obtain amplification at the good size).

But we don't observe results for L102(3), L102(6), L103(4), L106(1), L106(2), L106(4), L111(1)

Migration of an another gel for this sample...


Results:

PCR1_’’’L102(3;6))’’’ PCR2_’’’L103(4)’’’ PCR5_’’’L106(1; 2; 4)’’’ PCR10_’’’L111(1)’’’
Expected size Measured size Band Expected size Measured size Band Expected size Measured size Band Expected size Measured size Band
1045 pb 1000 pb 2-3 1003 pb - 4 1200 pb 1100 pb 5-6-7 1239 pb 1100 pb 8
Gel 13 : to solve Mistakes


==> Conclusion : We can observe a results for the samples : L102, L103, L106(4) and L111. (but not for L106(1; 2))

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