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Template:Team:KULeuven/3 September 2009/VanillinProduction
From 2009.igem.org
(Difference between revisions)
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| - | ===NOT PCR=== | + | ====NOT PCR==== |
* Electroporation of SAMS+TER ligation (from Tuesday ligation... the one that looked bad....) | * Electroporation of SAMS+TER ligation (from Tuesday ligation... the one that looked bad....) | ||
* Miniprepped the 4 colonies from SAMS | * Miniprepped the 4 colonies from SAMS | ||
| Line 17: | Line 17: | ||
|} | |} | ||
* Restriction digest of SAMS | * Restriction digest of SAMS | ||
| - | * Gel | + | * Gel electrophoresis of restriction from ''ech, fcs, sam5'' and ''sam8'' |
| - | ** | + | ** Result looks great (woohoo!) |
** Cut and purified from gel | ** Cut and purified from gel | ||
{| border ="1" align="center" | {| border ="1" align="center" | ||
!| gene || concentration || 260/280 || 260/230 | !| gene || concentration || 260/280 || 260/230 | ||
|- align="center" | |- align="center" | ||
| - | | | + | | ''sam5'' || 8,2 || 1,62 || 0,02 |
|- align="center" | |- align="center" | ||
| - | | | + | | ''sam8'' || 1,4 || 3,04 || 0,01 |
|- align="center" | |- align="center" | ||
| - | | ech || 10,1 || 1,50 || 0,05 | + | | ''ech'' || 10,1 || 1,50 || 0,05 |
|- align="center" | |- align="center" | ||
| - | | fcs || 5,2 || 1,61 || 0,03 | + | | ''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 | + | *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 ==== | ==== PCR ==== | ||
| Line 40: | Line 40: | ||
!| gene || concentration || 260/280 || 260/230 | !| gene || concentration || 260/280 || 260/230 | ||
|- align="center" | |- align="center" | ||
| - | | | + | | ''sam5'' || 361,6 || 1,91 || 2,13 |
|- align="center" | |- align="center" | ||
| - | | | + | | ''sam8'' || 323,8 || 1,92 || 2,06 |
|- align="center" | |- align="center" | ||
| - | | ech || 236,5 || 1,91 || 2,24 | + | | ''ech'' || 236,5 || 1,91 || 2,24 |
|- align="center" | |- align="center" | ||
| - | | fcs || 321,4 || 1,90 || 2,22 | + | | ''fcs'' || 321,4 || 1,90 || 2,22 |
|- | |- | ||
|} | |} | ||
| Line 53: | Line 53: | ||
!| gene || concentration || 260/280 || 260/230 | !| gene || concentration || 260/280 || 260/230 | ||
|- align="center" | |- align="center" | ||
| - | | | + | | ''sam5'' || 21,8 || 1,65 || 1,33 |
|- align="center" | |- align="center" | ||
| - | | | + | | ''sam8'' || 17,1 || 1,56 || 1,16 |
|- align="center" | |- align="center" | ||
| - | | ech || 13,8 || 1,78 || 1,46 | + | | ''ech'' || 13,8 || 1,78 || 1,46 |
|- align="center" | |- align="center" | ||
| - | | fcs || 20,9 || 1,70 || 0,20 | + | | ''fcs'' || 20,9 || 1,70 || 0,20 |
|- | |- | ||
|} | |} | ||
| - | * After the restriction, | + | * 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''. |
Latest revision as of 07:25, 18 September 2009
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.
