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From 2012.igem.org

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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