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 electrophoresis of restriction from ech, fcs, sam5 and sam8
- Result 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.
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