Team:Paris/July 29

From 2008.igem.org

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(→DNA digestion and purification)
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== '''Screening PCR of the transformations with Ligation '''==
 +
 +
Use of the same 8 clones of Biobricks that have been tested for cloning
 +
 +
 +
===Protocol===
 +
 +
* 50µl of Mix PCR by tubes
 +
* one 1 clone
 +
* Program : 105°C -  for 29 cycles : 95°C; 5' - 95°C; 30" - 55°C; 30" - 72°C; 1,5'
 +
 +
===Results===
 +
 +
* '''PCR 1 : J23109 (A)'''
 +
 +
{|border="1" Paris_PCR_1| '''PCRs setting''' (A)
 +
|
 +
|Quick Load
 +
|25µL
 +
|
 +
|
 +
|Expected size
 +
|-
 +
|Annealing
 +
|
 +
|n_oligoF
 +
|18 VF2
 +
|v_oligoF
 +
|10µM 1µL
 +
|1000pb
 +
|-
 +
|55°C
 +
|
 +
|n_oligoR
 +
|19 VR
 +
|v_oligoR
 +
|10µM 1µL
 +
|Sucess
 +
|-
 +
|Time Elongation
 +
|
 +
|Water
 +
|23µL
 +
|
 +
|
 +
|Yes
 +
|-
 +
|1m30'
 +
|
 +
|DNA
 +
|Toothpick of J23109
 +
|
 +
|
 +
|Image see bands 1 to 8
 +
|-
 +
|Number cycles
 +
|
 +
|
 +
|
 +
|
 +
|
 +
|[[Image:080717-1.png|thumb|J23109]]
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|-
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|29
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|
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|
 +
|
 +
|
 +
|
 +
|Ladder band 0
 +
|}
 +
 +
 +
* '''PCR 2 : E0030 (K)'''
 +
 +
{|border="1" Paris_PCR_2| Title = E0030 (K)
 +
|'''PCRs setting'''
 +
|
 +
|Quick Load
 +
|25µL
 +
|
 +
|
 +
|Expected size
 +
|-
 +
|Annealing
 +
|
 +
|n_oligoF
 +
|18 VF2
 +
|v_oligoF
 +
|10µM 1µL
 +
|723pb
 +
|-
 +
|55°C
 +
|
 +
|n_oligoR
 +
|19 VR
 +
|v_oligoR
 +
|10µM 1µL
 +
|Sucess
 +
|-
 +
|Time Elongation
 +
|
 +
|Water
 +
|23µL
 +
|
 +
|
 +
|Yes
 +
|-
 +
|1m30'
 +
|
 +
|DNA
 +
|Toothpick of E0030
 +
|
 +
|
 +
|Image see bands 9 to 16
 +
|-
 +
|Number cycles
 +
|
 +
|
 +
|
 +
|
 +
|
 +
|[[Image:080717-1.png|thumb|E0030]]
 +
|-
 +
|29
 +
|
 +
|
 +
|
 +
|
 +
|
 +
|Ladder band 0
 +
|}
 +
 +
 +
* '''PCR 3 : pSB1A3 (A)'''
 +
 +
{|border="1" Paris_PCR_3| Title = pSB1A3 (A)
 +
|'''PCRs setting'''
 +
|
 +
|Quick Load
 +
|25µL
 +
|
 +
|
 +
|Expected size
 +
|-
 +
|Annealing
 +
|
 +
|n_oligoF
 +
|18 VF2
 +
|v_oligoF
 +
|10µM 1µL
 +
|
 +
|-
 +
|55°C
 +
|
 +
|n_oligoR
 +
|19 VR
 +
|v_oligoR
 +
|10µM 1µL
 +
|Sucess
 +
|-
 +
|Time Elongation
 +
|
 +
|Water
 +
|23µL
 +
|
 +
|
 +
|Yes
 +
|-
 +
|1m30'
 +
|
 +
|DNA
 +
|Toothpick of pSB1A3
 +
|
 +
|
 +
|Image see bands 1 to 8
 +
|-
 +
|Number cycles
 +
|
 +
|
 +
|
 +
|
 +
|
 +
|[[Image:080717-2.png|thumb|pSB1A3]]
 +
|-
 +
|29
 +
|
 +
|
 +
|
 +
|
 +
|
 +
|Ladder band 0
|}
|}

Revision as of 09:24, 31 July 2008

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Contents

DNA digestion and purification

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)
  • 20,5 µL water

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).

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.

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




Screening PCR of the transformations with Ligation

Use of the same 8 clones of Biobricks that have been tested for cloning


Protocol

  • 50µl of Mix PCR by tubes
  • one 1 clone
  • Program : 105°C - for 29 cycles : 95°C; 5' - 95°C; 30" - 55°C; 30" - 72°C; 1,5'

Results

  • PCR 1 : J23109 (A)
Quick Load 25µL Expected size
Annealing n_oligoF 18 VF2 v_oligoF 10µM 1µL 1000pb
55°C n_oligoR 19 VR v_oligoR 10µM 1µL Sucess
Time Elongation Water 23µL Yes
1m30' DNA Toothpick of J23109 Image see bands 1 to 8
Number cycles
J23109
29 Ladder band 0


  • PCR 2 : E0030 (K)
PCRs setting Quick Load 25µL Expected size
Annealing n_oligoF 18 VF2 v_oligoF 10µM 1µL 723pb
55°C n_oligoR 19 VR v_oligoR 10µM 1µL Sucess
Time Elongation Water 23µL Yes
1m30' DNA Toothpick of E0030 Image see bands 9 to 16
Number cycles
E0030
29 Ladder band 0


  • PCR 3 : pSB1A3 (A)
PCRs setting Quick Load 25µL Expected size
Annealing n_oligoF 18 VF2 v_oligoF 10µM 1µL
55°C n_oligoR 19 VR v_oligoR 10µM 1µL Sucess
Time Elongation Water 23µL Yes
1m30' DNA Toothpick of pSB1A3 Image see bands 1 to 8
Number cycles
pSB1A3
29 Ladder band 0
Retrieved from "http://2008.igem.org/Team:Paris/July_29"