Team:TU Delft/2 August 2010 content
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
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 |