Team:Paris/July 29

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(Difference between revisions)
(→DNA digestion and purification)
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==DNA digestion and purification==
==DNA digestion and purification==
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for each reaction (total volume : 50 µL)
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=== Mix digestion ===
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''for each reaction (total volume : 50 µL)''
* 20 µL of DNA (MiniPrep product)
* 20 µL of DNA (MiniPrep product)
* 2 µL of enzyme 1
* 2 µL of enzyme 1
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* 20,5 µL water
* 20,5 µL water
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Each reaction was incubated 2 hours at 37°C, then 10 minutes at 60-65°C (to inactivate the enzymes). 10 µL of loading dye (6X) were added to each of the 50 µL of digestion product. The whole samples were run in a 1,5% agarose gel (about 30 minutes at 100 W ; 2 x 30 µL per sample ; 30 µL per well). The bands of interest were then excised from the gel and the DNA was purified using the QIAquick DNA Gel Extraction kit (QIAGEN). Unfortunately, there were not enough columms, so we took some columms from the QIAGEN MiniPrep kit, hoping that it will work with the QIAquick DNA Gel Extraction kit. Some of the samples were too voluminous, so we separated them into two tubes. The elution of DNA was performed using 50 µL of water (after 10 minutes of incubation at 37°C).
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 +
 
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=== Protocol ===
 +
 
 +
Each reaction was :
 +
* Incubate 2 hours at 37°C, then 10 minutes at 60-65°C (to inactivate the enzymes).  
 +
* Add 10 µL of loading dye (6X) to each of the 50 µL of digestion product.  
 +
* Run the whole samples in a '''1,5% agarose gel''' (about 30 minutes at 100 W ; 2 x 30 µL per sample ; 30 µL per well).  
 +
* Excise the bands of interest from the gel and the DNA was purified using the QIAquick DNA Gel Extraction kit (QIAGEN).  
 +
* The elution of DNA was performed using 50 µL of water (after 10 minutes of incubation at 37°C).
 +
 
 +
==> Unfortunately, there were not enough columms, so we took some columms from the QIAGEN MiniPrep kit, hoping that it will work with the QIAquick DNA Gel Extraction kit. Some of the samples were too voluminous, so we separated them into two tubes.  
 +
 
 +
 
Each of the samples was then analysed by a 1,5% agarose gel:
Each of the samples was then analysed by a 1,5% agarose gel:
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Results :  
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==> '''Conclusion :'''The amount of DNA loaded was too low and the DNA ladder used was the wrong one. So this experiment has to be reconducted tomorrow.
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The amount of DNA loaded was too low and the DNA ladder used was the wrong one. So this experiment has to be reconducted tomorrow.
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== '''PCR Screening of the Ligation Transformants'''==
== '''PCR Screening of the Ligation Transformants'''==

Revision as of 17:24, 4 August 2008

← Yesterday

↓ Calendar ↑

Tomorrow →

Contents

DNA digestion and purification

Mix digestion

for each reaction (total volume : 50 µL)

  • 20 µL of DNA (MiniPrep product)
  • 2 µL of enzyme 1
  • 2 µL of enzyme 2
  • 5 µL of buffer 2 (10X)
  • 0,5 µL of BSA
  • 20,5 µL water


Protocol

Each reaction was :

  • Incubate 2 hours at 37°C, then 10 minutes at 60-65°C (to inactivate the enzymes).
  • Add 10 µL of loading dye (6X) to each of the 50 µL of digestion product.
  • Run the whole samples in a 1,5% agarose gel (about 30 minutes at 100 W ; 2 x 30 µL per sample ; 30 µL per well).
  • Excise the bands of interest from the gel and the DNA was purified using the QIAquick DNA Gel Extraction kit (QIAGEN).
  • The elution of DNA was performed using 50 µL of water (after 10 minutes of incubation at 37°C).

==> Unfortunately, there were not enough columms, so we took some columms from the QIAGEN MiniPrep kit, hoping that it will work with the QIAquick DNA Gel Extraction kit. Some of the samples were too voluminous, so we separated them into two tubes.


Each of the samples was then analysed by a 1,5% agarose gel:

  • 2 µL of DNA
  • 3 µL of water
  • 1 µL of 6X loading dye

The ladder used was the 100 bp ladder from New England Biolabs.

KR000081.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 pb 10
D102 B0034 1 SpeI PstI BV 2077 pb 11
D105 R0079 1 SpeI PstI BV 2222 pb 13
D106 R0040 1 SpeI PstI BV 2119 pb 12
D108 S03154 1 XbaI PstI BI 707 pb 14
D111 S03879 1 XbaI PstI BI 725 pb 15
D115 C0179 2 EcoRI SpeI FI 746 pb 4 & 5
D116 C0179 2 XbaI PstI BI 745 pb 2 & 3
D117 E0030 1 EcoRI SpeI FI 746 pb 16
D128 B0030 2 EcoRI XbaI FV 2079 pb 6 & 7
D129 B0030 2 SpeI PstI BV 2080 pb 8 & 9

==> Conclusion :The amount of DNA loaded was too low and the DNA ladder used was the wrong one. So this experiment has to be reconducted tomorrow.

PCR Screening of the 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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