Team:TU Delft/6 September 2010 content
From 2010.igem.org
Labwork
Last week we did 2 DLTs, but both resulted in empty plates (no colonies). Apparently something went wrong, we think it was the diluted ligase that lost its activity. So, today we will try to do the DLT again, but then with a different ligase.
Digestion
# | Sample | Enzyme 1 | Enzyme 2 | Extra | Buffer | BSA |
1 | 018A | EcoRI | SpeI | AseI | 2 (Biolabs) | ✓ |
2 | 019A | EcoRI | XbaI | Phosphatase | 2 (Biolabs) | ✓ |
3 | J13002 | SpeI | PstI | Phosphatase | 2 (Biolabs) | ✓ |
4 | ladA | XbaI | PstI | AseI | 2 (Biolabs) | ✓ |
5 | 009A | EcoRI | SpeI | AseI | 2 (Biolabs) | ✓ |
6 | 010A | EcoRI | XbaI | Phosphatase | 2 (Biolabs) | ✓ |
7 | 104T | XbaI | PstI | ✗ | 2 (Biolabs) | ✓ |
8 | PalkB | SpeI | PstI | ✗ | 2 (Biolabs) | ✓ |
9 | 327 | SpeI | PstI | ✗ | 2 (Biolabs) | ✓ |
10 | K1 | XbaI | PstI | AseI | 2 (Biolabs) | ✓ |
11 | GFP-TT | XbaI | PstI | AseI | 2 (Biolabs) | ✓ |
Ligation
# | BioBrick | Fragment 1 | Recipient Vector/fragment 2 |
1 | 026 | E - J61101 - ADH - S (018) | E - pSB1A2 - J61107 - ALDH - X (019) |
2 | 027 | X - ladA - P | P - pSB1A2 - J13002 - S |
3 | 028 | E - J61100 - rubA4 - S (009) | X - J61100 - rubR - pSB1A2 - E (010) |
4 | 108 | X - BBC1 - B0015 - P | P - pSB1A2 - J13002 - S |
5 | 306 | X - K1 - P | P - pSB1A2 - PalkB - S |
6 | 331 | P - pSB1A2 - 327 - S | X - GFP-TT - P |
The ligation mixtures were ligated for 1 hour, after which they were added to competent K12 E.coli cells and plated on LB-agar with ampicillin.