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Template:Team:KULeuven/20 August 2009/VanillinProduction
From 2009.igem.org
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'''1)''' | '''1)''' | ||
| - | * EF colonies present! So, we miniprepped | + | * EF colonies present! So, we took 1ml off for further growth and miniprepped the remaining 4ml in triple |
* Nanodrop concentrations: | * Nanodrop concentrations: | ||
{| border ="1" align="center" | {| border ="1" align="center" | ||
| Line 13: | Line 13: | ||
* We performed a digest with EcoRI and SpeI to cut out the insert and check it's length. Enzymes were added separately, with 1h in between. Total volume was 30µl. | * We performed a digest with EcoRI and SpeI to cut out the insert and check it's length. Enzymes were added separately, with 1h in between. Total volume was 30µl. | ||
| - | * {| border ="1" align="center" | + | * Calculations restriction |
| + | {| border ="1" align="center" | ||
!| Part || µl DNA || µl MQ || | !| Part || µl DNA || µl MQ || | ||
|- align="center" | |- align="center" | ||
| Line 19: | Line 20: | ||
|- | |- | ||
|} | |} | ||
| + | |||
| + | * In the afternoon, the remaining 1ml was re-plated and put on 37°C | ||
'''2)''' | '''2)''' | ||
| - | * | + | * <u>Test 1</u>: Results from yesterday's restriction test were inconclusive, because the DNA ladder didn't run properly so the length of the fragments couldn't be read. Therefore, we did it again... |
* Restriction=OK! Test 1 completed. | * Restriction=OK! Test 1 completed. | ||
'''3)''' | '''3)''' | ||
| - | * | + | * <u>Test 2</u>: cutting with 2 enzymes subsequently (1h incubation in between) in a volume of 30µl. Using a larger volume should dilute the glycerol, which inhibits cutting. After that, the products are incubated overnight to allow full restriction. ''sam8'' and ''fcs'' are cut with EcoRI and SpeI; ''sam5'' and ''ech'' are cut with EcoRI and XbaI |
| - | *{| border ="1" align="center" | + | * Calculations restriction |
| + | {| border ="1" align="center" | ||
!| Part || µl DNA || µl MQ || | !| Part || µl DNA || µl MQ || | ||
|- align="center" | |- align="center" | ||
Latest revision as of 11:37, 14 September 2009
1)
- EF colonies present! So, we took 1ml off for further growth and miniprepped the remaining 4ml in triple
- Nanodrop concentrations:
| Part | concentration (ng/μl) | 260/280 λ | |
|---|---|---|---|
| EF A | 74,9 | 1,89 | |
| EF B | 63,1 | 1,87 | |
| EF C | 53,9 | 1,90 |
- We performed a digest with EcoRI and SpeI to cut out the insert and check it's length. Enzymes were added separately, with 1h in between. Total volume was 30µl.
- Calculations restriction
| Part | µl DNA | µl MQ | |
|---|---|---|---|
| EF C | 9,3 | 20,7 |
- In the afternoon, the remaining 1ml was re-plated and put on 37°C
2)
- Test 1: Results from yesterday's restriction test were inconclusive, because the DNA ladder didn't run properly so the length of the fragments couldn't be read. Therefore, we did it again...
- Restriction=OK! Test 1 completed.
3)
- Test 2: cutting with 2 enzymes subsequently (1h incubation in between) in a volume of 30µl. Using a larger volume should dilute the glycerol, which inhibits cutting. After that, the products are incubated overnight to allow full restriction. sam8 and fcs are cut with EcoRI and SpeI; sam5 and ech are cut with EcoRI and XbaI
- Calculations restriction
| Part | µl DNA | µl MQ | |
|---|---|---|---|
| Sam8 B | 7,9 | 22,1 | |
| Sam5 B | 4,0 | 26,0 | |
| Ech B | 5,2 | 24,8 | |
| Fcs B | 4,4 | 25,6 |
