Team:TU Delft/2 August 2010 content

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Revision as of 09:03, 4 August 2010

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.

TU Delft Pi2 Colony PCR part 1.png

TU Delft Pi2 Colony PCR part 2.png

Lane Description:

# Description Expected Length (bp) Primers Status Remarks
M SmartLadder n/a n/a n/a
1 BBa_K398305 3772 G00101 + G00100 X
2 BBa_K398305 3772 G00101 + G00100 X
3 BBa_K398305 3772 G00101 + G00100 X
4 BBa_K398305 3772 G00101 + G00100 X
5 BBa_K398305 3772 G00101 + G00100 X
6 BBa_K398101 1007 G00101 + G00100 X
7 BBa_K398101 1007 G00101 + G00100 X
8 BBa_K398101 1007 G00101 + G00100 X
m EZ Ladder n/a n/a n/a
M SmartLadder n/a n/a n/a

Emulsifier

When going over the plates in the fridge we noticed a few white colonies on plates from 13-07-'10. Previously we thought there were only red colonies. So we decided to do a colony PCR on all 12 white colonies.

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

1% Agarose of digestion check

The gel was runned at 100V for 1 hour, loaded 5 μL of marker and 5 μL sample + 1 μL loadingbuffer

Lane description:

# Description Expected Length (bp) Status
1 Smartladder
2 ‘E – J61100-AlkB2 – S’ (007T) ✗
3 Undigested 007T ✗
4 ‘E – J61100-rubA3 – X’ (008A) ✓
5 Undigested 008A ✓
6 ‘E – J61100-rubR – S’ (010A) ✓
7 Undigested 010A ✓
8 ‘E – B0015 – pSB1AK3 – X’ ✓
9 pSB1AK3-B0015 ✓
10 ‘E – J61100-ladA – S’ (017A) ✓
11 Undigested 017A ✓
12 ‘X – J61101-ADH – P’ (018A) ✓
13 Undigested 018A ✓
14 ‘E – J61107-ALDH – S’ (019A) ✓
15 Undigested (019A) ✓
16 ‘E – pSB1K3 – P’ ✓
17 Smartladder
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