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From 2008.igem.org

Digestion of DNA

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
D100	B0034	1	XbaI	PstI	BI	34 pb	-	0	8	5
		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

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