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From 2010.igem.org

Contents 1 Lab work 1.1 Alkane degradation 1.1.1 Digestion 1.2 Ligation

Lab work

Alkane degradation

BioBricks number 014K and 020A (the latter was already tried before but failed) are up today. We will do a digestion and a ligation followed by a transformation in order to make these BioBricks. Next to these two BioBricks we will also be working on placing some of the previously made BioBricks in the 1C3 plasmid so we can ship it to the registry.

Digestion

#	Sample	Enzyme 1	Enzyme 2	Enzyme 3	Buffer	BSA	Needed fragment
1	011A	EcoRI	SpeI	AseI	2 (Biolabs)	✓	‘E - J61100-alkB2-J61100-rubA3 - S’
2	013K	EcoRI	XbaI	✗	3 (Biolabs)	✓	‘E - J61100-rubA4-J61100-rubR-B0015-pSB1AK3 - X’
3	017A	EcoRI	SpeI	AseI	2 (Biolabs)	✓	‘E - J61100-ladA - S’
4	018A	EcoRI	XbaI	✗	3 (Biolabs)	✓	‘E - J61101-ADH-pSB1A2 - X’
5	011A	EcoRI	PstI	AseI	3 (Biolabs)	✗	‘E - J61100-alkB2-J61100-rubA3 - P’
6	013K	EcoRI	PstI	HindIII	2 (Biolabs)	✗	‘E - J61100-rubA4-J61100-rubR-B0015 - P’
7	021	EcoRI	PstI	SmaI	3 (Biolabs)	✓	‘E - J61107-ALDH-B0015 - P’
8	402	EcoRI	PstI	AseI	3 (Biolabs)	✗	‘E - J61101-PhPFDα - P’
9	403	EcoRI	PstI	AseI	3 (Biolabs)	✗	‘E - J61101-PhPFDβ - P’
10	pSB1C3	EcoRI	PstI	✗	3 (Biolabs)	✗	‘E - pSB1C3 - P’

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

[[Image:|300px|thumb|left|Digestion check]]

Ligation

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

#	BioBrick	Fragment 1	Fragment 2 / Plasmid
1	014K	‘E - J61100-alkB2-J61100-rubA3 - S’	‘E - J61100-rubA4-J61100-rubR-B0015-pSB1AK3 - X’
2	020A	‘E - J61100-ladA - S’	‘E - J61101-ADH-pSB1A2 - X’
3	011C	‘E - J61100-alkB2-J61100-rubA3 - P’	‘E - pSB1C3 - P’
4	013C	‘E - J61100-rubA4-J61100-rubR-B0015 - P’	‘E - pSB1C3 - P’
5	021C	‘E - J61107-ALDH-B0015 - P’	‘E - pSB1C3 - P’
5	402C	‘E - J61101-PhPFDα - P’	‘E - pSB1C3 - P’
5	403C	‘E - J61101-PhPFDβ - P’	‘E - pSB1C3 - P’

10 μL of this ligation mix were used to transform Top10 competent cells.