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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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== Lab work == | == 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''' | '''TuYV''' | ||
| Line 16: | Line 23: | ||
Wednesday: | 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 | + | *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, 1H and 2 plate. | + | 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: | Friday: | ||
| - | *Colonies from 1, 1H and 2 plate were picked and checked by colony PCR. But no bands were showed on the agrose gel | + | *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 | 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 | ||
| + | |||
---- | ---- | ||
[[https://2012.igem.org/Team:Wageningen_UR/Journal/week8 previous week]] [[https://2012.igem.org/Team:Wageningen_UR/Journal/week10 next week]] | [[https://2012.igem.org/Team:Wageningen_UR/Journal/week8 previous week]] [[https://2012.igem.org/Team:Wageningen_UR/Journal/week10 next week]] | ||
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
