Team:TU Delft/16 August 2010 content
From 2010.igem.org
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+ | |pAlkS cut | ||
+ | |1272, 1138, <b>106</b> | ||
+ | |12 | ||
|pSB1C3 cut | |pSB1C3 cut | ||
|<b>2035</b>, 1081 | |<b>2035</b>, 1081 |
Revision as of 19:07, 16 August 2010
Alkane degradation
Unfortunately last week's attempts hadn't yielded any BioBricks making use of the 2-way ligation method. We thus decided to take another stab at the 3-way ligation procedure, according to the BioBrick Assembly Manual of Ginkgo Bioworks. The BioBrick numbers we tried to construct were: 014C, 020C, 405C, 327C and 304C.
Digestion
# | Sample | Enzyme 1 | Enzyme 2 | Enzyme 3 | Buffer | BSA | Needed fragment |
1 | 011A | EcoRI | SpeI | AseI | NEBuffer 2 | ✓ | ‘E - J23100-J61100-alkB2-J61100-rubA3 - S’ |
2 | 013K | XbaI | PstI | HindIII | NEBuffer 2 | ✓ | ‘X - J61100-rubA4-J61100-rubR-B0015 - P’ |
3 | 017A | EcoRI | SpeI | AseI | NEBuffer 2 | ✓ | ‘E - J23100-J61100-ladA - S’ |
4 | 018A | XbaI | PstI | AseI | NEBuffer 2 | ✓ | ‘X - J61101-ADH - P’ |
5 | 402A | EcoRI | SpeI | AseI | NEBuffer 2 | ✓ | ‘E - J23100-J61101-PhPFDα - S’ |
6 | 403A | XbaI | PstI | AseI | NEBuffer 2 | ✓ | ‘X - J61101-PhPFDβ - P’ |
7 | pCaiF | EcoRI | SpeI | AseI | NEBuffer 2 | ✓ | ‘E - pCaiF - S’ |
8 | B0032 | XbaI | PstI | AseI | NEBuffer 2 | ✓ | ‘X - B0032 - P’ |
9 | B0032 | XbaI | PstI | AseI | NEBuffer 2 | ✓ | ‘X - B0032 - P’ |
10 | pAlkS | EcoRI | SpeI | AseI | NEBuffer 2 | ✓ | ‘E - pAlkS - P’ |
11 | pSB1C3 | EcoRI | PstI | None | NEBuffer 2 | ✓ | ‘E - pSB1C3 - P’ |
The digestions were executed for 15 minutes at 37 degrees, and checked on 1% agarose gel:
Lane description
# | Description | Expected size (bp) | OK? | |||
1 | Smartladder | Varies | Yes | |||
2 | 011A cut | 1523, 1199, 857 | Yes | |||
3 | 013K cut | 1825, 1592, 1342, 43 | Yes | |||
4 | 017A cut | 1410, 1199, 857 | Yes | |||
5 | 018A cut | 1185, 872, 793, 43 | Yes | |||
6 | 402A cut | 1199, 857, 543 | Yes | |||
7 | 403A cut | 1185, 872, 397, 43 | Yes | |||
8 | pCaiF in pANY cut | 1277, 1143, 74 | Yes | |||
9 | B0032 in pSB1A2 cut | 1185, 872, 35 | Yes | |||
10 | B0032 in pSB1A2 cut | 1185, 872, 35 | Yes | |||
11 | pAlkS cut | 1272, 1138, 106 | 12 | pSB1C3 cut | 2035, 1081 | Yes (However incomplete digestion, see 3116 bp) |
15 | BioRad EZ Load Marker | Varies | Yes |