Team:Paris/July 29

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.

Screening PCR of the transformations with Ligation

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

Protocol

• Mix

• 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

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
		2-->9			10-->17			2-->9			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
		2-->9			10-->17			2-->9			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
		2-->9			10-->17			2-->10			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
		2			3-->10			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
		2-->3			4-->11			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
		2-->4			5-->11			12-->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
		2-->5			6-->12			13-->16
Gel 10 : L121-L122-L125

PCR25_’’’L125(5-8)’’’			PCR26_’’’L1109.1(1-7)’’’			PCR27_’’’L109.2(1-8)’’’
Expected size	Measured size	Band	Expected size	Measured size	Band	Expected size	Measured size	Band
		2-->5			2-->9			10-->17
Gel 11 : L125			Gel 14 : L109.1-L109.2