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From 2010.igem.org

Alkane degradation

Yesterday's digestions showed that BioBrick 007 was not the expected length. From this it was concluded that the plasmid thus does not contain the desired insert, so we will make the BioBrick again. This was done, starting with a digestion:

#	Sample	Enzyme 1	Enzyme 2	Enzyme 3	Buffer	BSA	Needed fragment
1	J61100	SpeI	PstI	✗	2 (Biolabs)	✓	‘S – pSB1A2 - J61100– P’
2	1 μg alkB2	XbaI	PstI	AseI	2 (Biolabs)	✓	‘X – alkB2 – P’

The digestions were done during 2 hours, and checked on a gel:

Lane description

#	Description	Expected Length (bp)	Status
0	Smartladder (5μl)
1	‘S – pSB1A2 - J61100 – P’	2116
2	undigested pSB1A2-J61100
3	‘X – alkB2 – P’	1263
4	Undigested alkB2

Directly following the digestion, the products were ligated for 4 hours:

#	BioBrick	Fragment 1	Fragment 2
1	pSB1A2-J61100-alkB2	‘X – alkB2 – P’	‘S – pSB1A2-J61100 – P’