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From 2010.igem.org

Contents 1 Lab work 1.1 Tolerance and Sensing 1.1.1 Digestion, Ligation, Transformation 1.2 Alkane degradation

Lab work

Tolerance and Sensing

A new attempt to construct parts 304 (PalkS12 + B0032), 327(P(caif) + B0032) and 405 (402 + 403).

Digestion, Ligation, Transformation

1 microgram of each plasmid was digested:

#	Sample	Enzyme 1	Enzyme 2	Enzyme 3	Buffer	BSA	Needed fragment
1	PalkS12A	EcoRI	SpeI	ScaI	2 (Biolabs)	✓	‘E - PalkS12 - S’
2	P(caif)A	EcoRI	SpeI	ScaI	2 (Biolabs)	✓	‘E - P(caif) - S’
3	B0032	EcoRI	XbaI		2 (Biolabs)	✓	‘X - B0032 - E’
4	402: J61101 - PhPFDalpha	EcoRI	SpeI	ScaI	2 (Biolabs)	✓	‘E - J61101 - PhPFDalpha - S’
5	403: J61101 - PhPFDbeta	EcoRI	XbaI		2 (Biolabs)	✓	‘E - J61101 - PhPFDbeta - S’

The digestion was checked on a gel:

Lane description

#	Description	Expected size (bp)	OK?
1	Smartladder (5μl)	n/a	n/a
2	PalkS cut	106, 861, 1188	no
3	P(caif) cut	74, 861, 118	no
5	402 cut	543, 548, 1508	no
6	403 cut	2439, 58	vague

So most digestions were not what we expected. The ligations were already started anyway.

Following the digestions, the fragments were ligated for 4 hours.

#	BioBrick	Fragment 1	Recipient vector
1	304A	12.5 μL ‘E - PalkS12 - S’	10 μL ‘X - B0032 - E’
2	327A	10 μL ‘E - P(caif) – S’	10 μL ‘X - B0032 - E’
3	405A	15 μL ‘E - J61101 - PhPFDalpha – S’	3 μL ‘X - J61101 - PhPFDbeta - E’

Alkane degradation

All of yesterday's plates contained colonies. To check whether they contain the correct insert we will do a colony PCR.

Lane description

#	Description	Primers	Expected length (bp)	✓	✗
L	SmartLadder	n/a	n/a	n/a	n/a
1-5	013A	G00100 + G00101	1929	none	all
6-10	013K	G00100 + G00101	1929	9	6,7,8,10
11-15	020A	G00100 + G00101	2482	none	all
L	SmartLadder	n/a	n/a	n/a	n/a

On the gel it looks like lane 9, containing 013K, has the correct length. We will do a plasmid isolation tomorrow. Unfortunately none of the colonies of 020A seemed to be correct. We will check some more colonies with a colony PCR overnight.