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Template:Team:KULeuven/3 September 2009/VanillinProduction
From 2009.igem.org
NOT PCR
- Electroporation of SAMS+TER ligation (from Tuesday ligation... the one that looked bad....)
- Miniprepped the 4 colonies from SAMS
| part | concentration | 260/280 | 260/230 |
|---|---|---|---|
| SAMS 1 | 35,4 | 1,97 | 1,18 |
| SAMS 2 | 49,3 | 2,00 | 0,97 |
| SAMS 3 | 62,0 | 1,97 | 2,65 |
| SAMS 4 | 78,3 | 1,85 | 1,43 |
| Terminator | 181,4 |
- Restriction digest of SAMS
- Gel electrophorese of restriction from ech, fcs, sam5 and sam8
- Results looks great (woohoo!)
- Cut and purified from gel
| gene | concentration | 260/280 | 260/230 |
|---|---|---|---|
| Sam5 | 8,2 | 1,62 | 0,02 |
| Sam8 | 1,4 | 3,04 | 0,01 |
| ech | 10,1 | 1,50 | 0,05 |
| fcs | 5,2 | 1,61 | 0,03 |
- Used ech and fcs to ligate using 5 μl from ech and 23,5 μl from fcs. There was not enough Sam8 for ligation. Redo restriction at some point in the future...
- 4 colonies from Sam8 and ech were picked and plated
PCR
- The PCR from yesterday went very well, we had a huge amount of amplified biobrick DNA of the different parts. Agarose gel electrophoresis was used to test if the different pieces of DNA created by PCR had the correct length.
| gene | concentration | 260/280 | 260/230 |
|---|---|---|---|
| Sam5 | 361,6 | 1,91 | 2,13 |
| Sam8 | 323,8 | 1,92 | 2,06 |
| ech | 236,5 | 1,91 | 2,24 |
| fcs | 321,4 | 1,90 | 2,22 |
- Amplified DNA was purified and cut with different restriction enzymes. After restriction, DNA was purified again before ligation.
| gene | concentration | 260/280 | 260/230 |
|---|---|---|---|
| Sam5 | 21,8 | 1,65 | 1,33 |
| Sam8 | 17,1 | 1,56 | 1,16 |
| ech | 13,8 | 1,78 | 1,46 |
| fcs | 20,9 | 1,70 | 0,20 |
- After the restriction, Sam5, Sam8 and terminator were ligated in a three-way ligation. The same was done for ech, fcs and terminator. We also started a ligation of just Sam5 and Sam8 and ech and fcs.
