"

From 2010.igem.org

Alkane Sensing, Solvent Tolerance and Salt Tolerance

by Pieter

The plates containing yesterday's ligations contained colonies, to check whether they really contain the desired BioBrick a colony PCR was done, and the used colonies were grown in liquid LB medium over night. The results from the PCR were analysed on a 1% agarose gel.

Alkane degradation

For the next step in BioBrick formation a digestion was done:

#	Sample	Enzyme 1	Enzyme 2	Enzyme 3	Buffer	BSA	Needed fragment
1	1 μg 007T	EcoRI	SpeI	BamH1	2 (Biolabs)	✓	‘E – J61100-AlkB2 – S’
2	1 μg 008A	EcoRI	XbaI		2 (Biolabs)	✓	‘E – J61100-rubA3 – X’
3	1 μg 010A	EcoRI	SpeI	AseI	2 (Biolabs)	✓	‘E – J61100-rubR – S’
4	2 μg B0015	EcoRI	XbaI		2 (Biolabs)	✓	‘E – B0015 – pSB1AK3 – X’
5	1 μg 017A	EcoRI	SpeI	AseI	2 (Biolabs)	✓	‘E – J61100-ladA – S’
6	1 μg 018A	XbaI	PstI	AseI	2 (Biolabs)	✓	‘X – J61101-ADH – P’
7	1 μg 019A	EcoRI	SpeI	AseI	2 (Biolabs)	✓	‘E – J61107-ALDH – S’
8	1 μg pSB1K3	EcoRI	PstI		2 (Biolabs)	✓	‘E – pSB1K3 – P’

Results of the digestion on 1% agarose gel gel runned at 100V for 1 hour loaded 5 μL of marker and 10 μL sample + 2 μL loadingbuffer

Lane description:

#	Description	Expected Length (bp)	Status	Remarks'
	marker
1	Undigested part name + vector (e.g. B0015 in pSB1AK3)		✓or ✗
2	Part name + vector (e.g. B0015 in pSB1AK3) + Enzyme 1 + Enzyme 2		✓or ✗
3	Undigested part		✓or ✗
4	Part + Enzyme 1 + Enzyme 2		✓or ✗
5	Undigested part		✓or ✗
6	Part + Enzyme 1 + Enzyme 2		✓or ✗
7	Undigested part		✓or ✗
8	Part + Enzyme 1 + Enzyme 2		✓or ✗
9	Undigested part		✓or ✗
10	Part + Enzyme 1 + Enzyme 2		✓or ✗
11	Undigested part		✓or ✗
12	Part + Enzyme 1 + Enzyme 2		✓or ✗
13	Undigested part		✓or ✗
14	Part + Enzyme 1 + Enzyme 2		✓or ✗
15	Undigested part		✓or ✗
16	Part + Enzyme 1 + Enzyme 2		✓or ✗
17	Undigested part		✓or ✗
18	Part + Enzyme 1 + Enzyme 2		✓or ✗
19	Undigested part		✓or ✗
20	Part + Enzyme 1 + Enzyme 2		✓or ✗