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From 2010.igem.org

Contents 1 Lab work 1.1 Ordered DNA 1.2 Solvent Tolerance and Hydrocarbon Sensing 1.3 Emulsifier 1.4 Characterization of Anderson RBS sequences

Lab work

Ordered DNA

We have now stock of the ordered DNA, to make a real BioBrick of this DNA we are going to ligated it into the iGEM plasmid backbone SB1C3. First we digested the plasmids:

#	Digestion reaction	Used Buffer	Needed fragment
1	1 μg alkB2+ EcoRI + PstI	Buffer 3 (BioLabs)	‘E – alkB2– P’
2	1 μg rubA3+ SpeI + PstI	Buffer 3 (BioLabs)	‘E – rubA3– P’
3	1 μg ladA + SpeI + PstI	Buffer 3 (BioLabs)	‘E – ladA – P’
4	1 μg ADH + SpeI + PstI	Buffer 3 (BioLabs)	‘E – ADH – P’
5	1 μg AlnA + SpeI + PstI	Buffer 3 (BioLabs)	‘E – AlnA – P’
6	1 μg OprG + SpeI + PstI	Buffer 3 (BioLabs)	‘E – OprG – P’
7	1 μg AlkS + SpeI + PstI	Buffer 3 (BioLabs)	‘E – AlkS – P’
8	1 μg PalkB + SpeI + PstI	Buffer 3 (BioLabs)	‘E – PalkB – P’
9	1 μg PalkS12+ SpeI + PstI	Buffer 3 (BioLabs)	‘E – PalkS12– P’
10	1 μg PhPFDα + SpeI + PstI	Buffer 3 (BioLabs)	‘E – PhPFDα – P’
11	1 μg PhPFDβ + SpeI + PstI	Buffer 3 (BioLabs)	‘E – PhPFDβ – P’
12	3 μg pSB1C3 + EcoRI + PstI	Buffer 3 (BioLabs)	‘E – ---- – P’

Solvent Tolerance and Hydrocarbon Sensing

Some BioBricks are in production. Digestion reaction

#	Digestion reaction	Used Buffer	Needed fragment
1	1 μg PhPFDα + EcoRI + SpeI	Buffer 2 + BSA (BioLabs)	‘E – PhPFDα – S’
2	1 μg PhPFDβ + EcoRI + SpeI	Buffer 2 + BSA (BioLabs)	‘E – PhPFDβ – S’
3	1 μg AlkS + EcoRI + SpeI	Buffer 2 + BSA (BioLabs)	‘E – AlkS – S’
4	1 μg PalkB + EcoRI + SpeI	Buffer 2 + BSA (BioLabs)	‘E – PalkB – S’
5	1 μg B0032 + XbaI + PstI	Buffer 2 + BSA (BioLabs)	‘X – B0032 – P’
6	1 μg B0015 + XbaI + PstI	Buffer 2 + BSA (BioLabs)	‘X – B0015 – P’
7	1 μg E0422 + XbaI + PstI	Buffer 2 + BSA (BioLabs)	‘X – E0422 – P’
8	3 μg pSB1T3 + EcoRI + PstI	Buffer 2 + BSA (BioLabs)	‘E - ---- - P’

Emulsifier

Today the digestion products from yesterday were run on gel to see whether the plasmids were cut in the right way.

Lane description

#	Description	Expected Length (bp)
1	Biorad EZ marker (5 μL)
2	Undigested pSB1T3 (10 μL + 2 μL loadingbuffer)
3	Digested pSB1T3 (10 μL + 2 μL loadingbuffer)	2500
4	Undigested AlnA (10 μL + 2 μL loadingbuffer)
5	Digested AlnA (10 μL + 2 μL loadingbuffer)	1107
6	Undigested OprG (10 μL + 2 μL loadingbuffer)
7	Digested OprG (10 μL + 2 μL loadingbuffer)	744
8	Undigested B0015 (10 μL + 2 μL loadingbuffer)
9	Digested B0015 (10 μL + 2 μL loadingbuffer)	130
10	Undigested R0011 (10 μL + 2 μL loadingbuffer) !
11	Digested R0011 (10 μL + 2 μL loadingbuffer)	55
12	Undigested B0032 (10 μL + 2 μL loadingbuffer)
13	Digested B0032 (10 μL + 2 μL loadingbuffer)	13
14	SmartLadder (5 μL)

! Sample not fully loaded on gel

Characterization of Anderson RBS sequences