Team:Wageningen UR/Journal/week9

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= week 9: 25 june - 1 july =
= week 9: 25 june - 1 july =
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== Office work ==
 
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'''General'''
'''General'''
-
cloning of BBa_J04500 (IPTG inducible promotor with RBS) and BBa_K197022/38 (Berkeley KILR coil)
+
Cloning of BBa_J04500 (IPTG inducible promotor with RBS) and BBa_K197022/38 (Berkeley KILR coil)
-
*BBa_J04500 was solubilized from the Standard registry parts plate I
+
*BBa_J04500 was solubilized from the Standard registry parts plate I with 10µl MQ water
 +
*BBa_K197022/38 was ordered from the registry
* transformation of ''E.coli'' with both plasmids
* transformation of ''E.coli'' with both plasmids
*5 samples (due to variation of transformation protocol)
*5 samples (due to variation of transformation protocol)
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Written by: Han Yue
Written by: Han Yue
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 +
'''CCMV'''
 +
*Colony PCR of all CCMV variants
 +
-Programme:
 +
  10 min 95 C
 +
  30 sec 95 C |
 +
  30 sec 60 C | x 35
 +
  1  min 72 C |
 +
  10 min 72 C
 +
  store at 4 C
 +
-PCR reaction mixture
 +
  2 ul  Buffer
 +
  0.4ul dNTPs
 +
  0.4ul FWD primer
 +
  0.4ul RVS primer
 +
  0.1ul Enzyme (taq)
 +
16.7ul H20
 +
 +
*Ligated CCMV fragment into pJET for better digestion, transformed E.coli with the pJET fragment
 +
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Latest revision as of 23:00, 26 September 2012

week 9: 25 june - 1 july

Lab work

General

Cloning of BBa_J04500 (IPTG inducible promotor with RBS) and BBa_K197022/38 (Berkeley KILR coil)

  • BBa_J04500 was solubilized from the Standard registry parts plate I with 10µl MQ water
  • BBa_K197022/38 was ordered from the registry
  • transformation of E.coli with both plasmids
  • 5 samples (due to variation of transformation protocol)
  • plated on LB-agar with corresponding antibiotic
  • 5 colonies picked per plate and grown in 5 ml liquid LB overnight
  • subsequent miniprep and digestion check
  • electrophoresis gel showed the expected bands after 20 min running, unfortunately DNA got bleached later by UV light

3 glycerol stocks (25%) of samples 1,3 and 5 were made to test the protocol for freezing cells at 80°C and subsequent thawing and streaking out, which was done successfully the next day.

TuYV

Wednesday:

  • Last week's PCR products(1,1H,2,2H,3,3H) were digested, ligated into the linearized pSB1A3 backbone and transformed into our electrocompetent DH5-alpha cells.

Then after transformation, we checked the colonies on the plate, but only few colonies on positive control and sample 1(TuYV coat protein), 1H(TuYV coat protein his-tag) and 2 plate (TuYV coat protein + 1/2 readthrough).


Friday:

  • Colonies from 1, 1H and 2 plate were picked and checked by colony PCR. But no bands were showed later on the agrose gel check.

Written by: Han Yue

CCMV

  • Colony PCR of all CCMV variants

-Programme: 

  10 min 95 C
  30 sec 95 C |
  30 sec 60 C | x 35
  1  min 72 C |
  10 min 72 C
  store at 4 C

-PCR reaction mixture 

 2 ul  Buffer
 0.4ul dNTPs
 0.4ul FWD primer
 0.4ul RVS primer
 0.1ul Enzyme (taq)
16.7ul H20
  • Ligated CCMV fragment into pJET for better digestion, transformed E.coli with the pJET fragment


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