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	|'''#'''		|'''#'''
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	'''Ligation'''		'''Ligation'''
-	Ligation over night at 16 C.	+	The digestion products were [[Team:TU_Delft/protocols/ligation|ligated]] over night:
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		+	|'''#'''
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		+	|5 μL Cut plasmid with B0032 + 10 μL PCR Product of R0011
		+	|}

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Revision as of 14:14, 19 July 2010

Lab work

Alkane degradation

Biobricks in production:

Digestion reactions:

#	Digestion reaction	Used Buffer	Needed fragment
1	2 μg J61100 + EcoRI + SpeI	NEBuffer 2	‘E – J61100 – S’
2	2 μg J61100 + EcoRI + SpeI	NEBuffer 2	‘E – J61100 – S’
3	1 μg rubR + EcoRI + SpeI	NEBuffer 2	‘E – rubR– S’
4	1 μg J61101 + EcoRI + SpeI	NEBuffer 2	‘E – J61101– S’
5	1 μg J61107 + EcoRI + SpeI	NEBuffer 2	‘E – J61107– S’
6	1μg alkB2 + Xbal + PstI	NEBuffer 2	‘X – alkB2 – P’
7	1 μg rubA3 + Xbal + PstI	NEBuffer 2	‘X – rubA3 – P’
8	1 μg rubA4 + Xbal + PstI	NEBuffer 2	‘X – rubA4 – P’
9	1 μg B0015 + Xbal + PstI	NEBuffer 2	‘X – B0015 – P’
10	1 μg ladA + Xbal + PstI	NEBuffer 2	‘X – ladA – P’
11	1 μg ADH + Xbal + PstI	NEBuffer 2	‘X – ADH – P’
12	1 μg ALDH + Xbal + PstI	NEBuffer 2	‘X – ALDH – P’
13	3 μg pSB1T3 + EcoRI + PstI	NEBuffer 2	‘E – pSB1T3(lin) – P’

Emulsifier

Since last weeks attempt to construct the inducible protomer R0011 with RBS B0032 failed, we decided to take a different approach. Instead of doing the standard 2 parts assembly, we amplify the promoter by PCR digest it and ligate in the plasmid that contains the RBS which has only be cut open at the left side. By doing so we do not risk losing the super small DNA fragments like the RBS. The problem is that we cannot use antibiotics selection. So we need to determine that by Colony PCR and sequencing.

PCR Amplification

[http://partsregistry.org/Part:BBa_R0011 R0011] was amplified with the universal primers. The product was put on gel:

Lane description:

#	Description	Amount
1	BioRad EZ Ladder	5 ul
2	PCR Product	10 ul + 2 ul LB

The PCR band is about 300 bps long. R0011 itself is just 55 bp, but the flanking primer regions are about 100 bp each.

Digestion

The PCR product of promotor R0011 was cut with ... and the RBS containing plasmid has been cut open with ... at 37 C.

Ligation

The digestion products were ligated over night:

#	Ligation reaction
1	5 μL Cut plasmid with B0032 + 10 μL PCR Product of R0011