Template:Team:KULeuven/20 August 2009/VanillinProduction

From 2009.igem.org

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* EF colonies present! So, we miniprepped them in triple
+
'''1)'''
 +
* 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"
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|}
|}
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* 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
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{| border ="1" align="center"
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!| Part || µl DNA || µl MQ ||
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|- align="center"
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| EF C ||9,3 || 20,7 || 
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|-
 +
|} 
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* 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...
+
* In the afternoon, the remaining 1ml was re-plated and put on 37°C
 +
 
 +
 
 +
'''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.
 +
 
 +
 
 +
'''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
 +
 
 +
* Calculations restriction
 +
{| border ="1" align="center"
 +
!| Part || µl DNA || µl MQ ||
 +
|- align="center"
 +
| Sam8 B ||7,9 || 22,1 ||
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|- align="center"
 +
| Sam5 B ||4,0 || 26,0 || 
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|- align="center"
 +
| Ech B ||5,2 || 24,8 ||
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|- align="center"
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| Fcs B ||4,4 || 25,6 ||
 +
|-
 +
|}

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
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