Team:TU Delft/13 July 2010 content

From 2010.igem.org

(Difference between revisions)
(Lab work)
(Characterization of Anderson RBS sequences)
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'''!''' Sample not fully loaded on gel
'''!''' Sample not fully loaded on gel
==Characterization of Anderson RBS sequences==
==Characterization of Anderson RBS sequences==
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The band position of the digestion products of AlnA and OprG correspond to their length. The other fragments (R0011, B0032 and B0015) probably run of the gel.
 
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The ligation products that were incubated over night were transformed to Top10 competent cells according to the [[Team:TU_Delft/protocols/transformation|protocol]].
 

Revision as of 19:50, 19 July 2010

Contents

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.

1% Agarose gel runned at 100V for 1h
EuroGentec SmartLadder

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