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From 2010.igem.org

Contents 1 Lab work 1.1 Alkane Degradation 1.1.1 PCR Amplification 1.1.2 Plasmid isolation

Lab work

Alkane Degradation

PCR Amplification

K398007A, K398008A, K398009A, K398010A, K398017A, K398018A, K398019A, B0015 were amplified with the universal primers G00100 and G00101. The product was put on 1% agarose gel:

Lane Description:

#	Description	Expected Length (bp)	Primers	Status	Remarks
M1	SmartLadder	n/a	n/a	n/a
1	PCR product of 007	1616	G00101 + G00101	✓
2	PCR product of 008	473	G00101 + G00101	✓
3	PCR product of 009	482	G00101 + G00101	✓
4	PCR product of 010	1505	G00101 + G00101	✓
5	PCR product of 017	1625	G00101 + G00101	✓
6	PCR product of 018	1052	G00101 + G00101	✓
7	PCR product of 019	1796	G00101 + G00101	✓
8	PCR product of B0015	445	G00101 + G00101	✓

The PCR products were subsequently digested:

#	Sample	Enzyme 1	Enzyme 2	Buffer	BSA	Needed fragment
1	40 μL PCR product 007	EcoRI	SpeI	2 (BioLabs)	✓	‘E–007–S’
2	40 μL PCR product 008	XbaI	PstI	2 (BioLabs)	✓	‘X–008-P’
3	40 μL PCR product 009	EcoRI	SpeI	2 (BioLabs)	✓	‘E–009–S’
4	40 μL PCR product 010	XbaI	PstI	2 (BioLabs)	✓	‘X–010-P’
5	40 μL PCR product 017	EcoRI	SpeI	2 (BioLabs)	✓	‘E–009–S’
6	40 μL PCR product 018	XbaI	PstI	2 (BioLabs)	✓	‘X–018-P’
7	40 μL PCR product 019	EcoRI	SpeI	2 (BioLabs)	✓	‘E–019-S’
8	40 μL PCR product B0015	XbaI	PstI	2 (BioLabs)	✓	‘X–B0015-P’

The digestion products were ligated overnight:

#	BioBrick	Fragment 1	Fragment 2	Final volume
1		4 μL ‘E–007–S’	4 μL ‘X–00–P’
2		? μL ‘E – Part – S’	? μL ‘X – Part – P’
3		? μL ‘E – Part – S’	? μL ‘X – Part – P’
4		? μL ‘E – Part – S’	? μL ‘X – Part – P’
5		? μL ‘E-Part1-S’	? μL ‘X-Part2-P'
6		? μL ‘E – Part – S’	? μL ‘X – Part – P’
7		? μL ‘E – Part – S’	? μL ‘X – Part – P’
8		? μL ‘E – Part – S’	? μL ‘X – Part – P’

Plasmid isolation

A plasmid isolation was done on the positive colonies of yesterday. This was done using a QIAgen Miniprep kit.