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:

1% agarose of PCR. Gel runned at 100V for 1 hour. Of all samples 10 μL + 2 μL loadingbuffer was loaded. 5 μL was loaded of marker

Lane Description:

#	Description	Expected Length (bp)	Primers	Status
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	✓

Digestion

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’

Ligation

The digestion products were ligated overnight:

#	BioBrick	Fragment 1	Fragment 2	Final volume
1	K398011	4 μL ‘E–007–S’	4 μL ‘X–008–P’	10 μL
2	K398012	4 μL ‘E–009–S’	4 μL ‘X–010–P’	10 μL
3	K398020	4 μL ‘E–017–S’	4 μL ‘X–018–P’	10 μL
4	K398021	4 μL ‘E–019–S’	4 μL ‘X–B0015–P’	10 μL
5	negative control	4 μL ‘E–007–S’	4 μL H2O	10 μL

Plasmid isolation

A plasmid isolation was done on the positive colonies of yesterday. This was done using a Qiagen Miniprep kit. We used 2 mL of the bacterial cells to make -80 °C stocks.

Team:TU Delft/29 July 2010 content

From 2010.igem.org

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