Team:Paris/July 29

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(DNA digestion and purification)
(DNA digestion and purification)
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Revision as of 21:37, 30 July 2008

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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 - 10
2 - - -
D101 B0034 3 EcoRI XbaI FV 2076 pb - - 1 & 7 2 & 2
D102 B0034 4 SpeI PstI BV 2077 pb 2000 pb 10 1 & 7 3 & 3
5 - 1 & 7 4 & 4
D103 J23101 1 SpeI PstI BV 2100 pb 2000 pb 10 1 5
2 - 1 6
D104 J23109 1 SpeI PstI BV 2100 pb 1 7
2 1 8
D105 R0079 1 SpeI PstI BV 2222 pb 2 2
2 2 3
D106 R0040 1 SpeI PstI BV 2119 pb 2 & 7 4 & 5
2 2 & 7 5 & 6
D107 S03154 1 SpeI PstI BV 2750 pb 2 & 7 6 & 7
D108 S03154 2 XbaI PstI BI 707 pb 2 & 7 7 & 8
3 2 & 7 8 & 9
D109 S03154 4 EcoRI SpeI FI 708 pb 3 & 7 2 & 10
D110 S03879 1 SpeI PstI BV 2768 pb 3 3