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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

Friday, a colony PCR on colonies from a plate with transformants with BioBrick 013AK did not give positive results. But this doesn't mean that none of the colonies are correct. To check we did another colony PCR today on 8 more colonies of the AMP plate as well as the KAN plate. Hopefully a the PCR product of a colony will show the correct length.