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From 2010.igem.org

Contents 1 Alkane degradation 1.1 Digestion, Ligation, Transformation 1.1.1 Ligation 1.2 Colony PCR

Alkane degradation

Digestion, Ligation, Transformation

A digestion was done on a number of BioBricks for the production of new BioBricks.

#	Sample	Enzyme 1	Enzyme 2	Enzyme 3	Buffer	BSA	Needed fragment
1	009A	EcoRI	SpeI	PvuI	2 (Biolabs)	✓	‘E - J61100 - rubA4 - S’
2	012K	EcoRI	XbaI	✗	2 (Biolabs)	✓	‘E – J61100 - rubR - B0015 – X’
3	017A	SpeI	PstI		2 (Biolabs)	✓	‘P - pSB1A2 – J61100 - ladA – S’
4	018A	XbaI	PstI	PvuI	3 (Biolabs)	✓	‘X – J61107 - ALDH – P’

The digestion was checked on a gel, together with the uncut BioBricks:

Lane description

#	Description	Expected size (bp)	OK?	#	Description	Expected size (bp)	OK?
L	Smartladder (3μl)	n/a	n/a	5	017A cut
1	009A cut			6	017A uncut	n/a
2	009A uncut	n/a		7	018A cut
3	012K cut			8	018A uncut	n/a
4	012K uncut	n/a

Ligation

Following the digestions, the fragments were [Team:TU_Delft/protocols/ligation ligated]] for 4 hours.

#	BioBrick	Fragment 1	Recipient vector
1	013AK	10 μL ‘E – J61100 - rubA4 – S’	10 μL ‘X – J61100 - rubR - B0015 - pSB1AK3 – E’
2	020A	10 μL ‘P - pSB1A2 – J61100 - ladA – S’	10 μL ‘X – J61107 - ALDH – P’

Colony PCR