Team:Lethbridge/Notebook/Lab Work/Group3 - 2011.igem.org
Team:Lethbridge/Notebook/Lab Work/Group3
From 2011.igem.org
(Difference between revisions)
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| | 3. [[Team:Lethbridge/Notebook|Transformation]] | | 3. [[Team:Lethbridge/Notebook|Transformation]] |
| - | * Transformed lumazine synthase - dt in pSB1C3 into DH5∝ | + | * Transformed lumazine synthase - dt in pSB1C3 into DH5∝ E. coli cells |
| - | * Transformed pBAD-RBS in pSB1C3 into DH5∝ | + | * Transformed pBAD-RBS in pSB1C3 into DH5∝ E. coli cells |
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| | ==June 24th, 2011== | | ==June 24th, 2011== |
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| | Ran on 1% TAE agarose gel for 90 minutes at 80V <br /> | | Ran on 1% TAE agarose gel for 90 minutes at 80V <br /> |
| - | * Gel melted | + | * Gel melted -- unsuccessful. |
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| | colonies picked see lab book team 3/ team 1 pg.42 | | colonies picked see lab book team 3/ team 1 pg.42 |
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| | ==August 4th, 2011== | | ==August 4th, 2011== |
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| - | [[Team:Lethbridge/Notebook|Restriction]] | + | [[Team:Lethbridge/Notebook|Restriction]] ( |
| | * Enhanced lumazine synthase restricted with EcoR1 and Pst1 | | * Enhanced lumazine synthase restricted with EcoR1 and Pst1 |
| | * pSB1C3 restricted with EcoR1 and Pst1 | | * pSB1C3 restricted with EcoR1 and Pst1 |
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| | * lumazine - dt-pBAD -RBS restricted with EcoR1 and Pst1 | | * lumazine - dt-pBAD -RBS restricted with EcoR1 and Pst1 |
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| - | Gel from August 2nd re-done | + | Gel ran at 80V for 90 minutes |
| - | * Ran at 80V for 90 minutes
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| | [[Team:Lethbridge/Notebook|Gel Extraction]] performed | | [[Team:Lethbridge/Notebook|Gel Extraction]] performed |
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| | * lumazine - dt -pBAD - RBS with pSB1C3 | | * lumazine - dt -pBAD - RBS with pSB1C3 |
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| - | [[Team:Lethbridge/Notebook|Ligation]] | + | [[Team:Lethbridge/Notebook|Transformation]] |
| | + | * Transformation of DNA into competent DH5∝ E. coli cells |
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| | + | ==August 5th, 2011== |
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| | + | Transfer of p0440 - K331033 from pSB1A2 into pSB1C3 |
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| | + | [[Team:Lethbridge/Notebook|Restriction]] |
| | + | * p0440 - K331033 restricted with EcoR1 and Pst1 |
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| | + | Ran on 1% TAE gel for 90 minutes at 80V |
| | + | * Unsuccessful restriction |
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| | + | ==August 6th, 2011== |
| | + | |
| | + | Transfer of p0440 - K331033 from pSB1A2 into pSB1C3 |
| | + | |
| | + | [[Team:Lethbridge/Notebook|Restriction]] |
| | + | * p0440 - K331033 restricted with EcoR1 and Pst1 |
| | + | |
| | + | Ran on 1% TAE gel for 90 minutes at 80V |
| | + | *Gel melted -- unsuccessful |
Revision as of 03:40, 15 September 2011
Contents
- 1 June
- 1.1 June 21st, 2011
- 1.2 June 24th, 2011
- 1.3 June 25th, 2011
- 1.4 June 28th
- 1.5 July 8th
- 1.6 July 10th, 2011
- 1.7 July 25th, 2011
- 1.8 July 28th, 2011
- 1.9 July 29th, 2011
- 1.10 August 2nd, 2011
- 1.11 August 4th, 2011
- 1.12 August 5th, 2011
- 1.13 August 6th, 2011
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June
June 21st, 2011
Restriction, Ligation, and Transformation
Lumazine synthase - double terminator and pBAD - RBS into pSB1C3
1. Restriction
- Restricted lumazine synthase - dt out of pSB1K3 and pBAD - RBS out of pSB1T3 with EcoR1 and Pst1
- Restricted destination plasmid pSB1C3 with EcoR1 and Pst1
2. Ligation
- Ligated lumazine synthase-dt into destination plasmid pSB1C3
- Ligated pBAD-RBS into destination plasmid pSB1C3
3. Transformation
- Transformed lumazine synthase - dt in pSB1C3 into DH5∝ E. coli cells
- Transformed pBAD-RBS in pSB1C3 into DH5∝ E. coli cells
June 24th, 2011
Picked cells from June 21st transformation
June 25th, 2011
Minipreped Cells picked on June 25th, 2011
June 28th
Restriction digest and ran on gel
Lumazine - dt construct successfully inserted into pSB1C3
Glycerol stocked.
Add Gel Picture
July 8th
Performed antibiotic assay on lumazine - dt in pSB1C3
Samples from one colony grew in Kanamicyn; this stock was discarded
PCR
- PCR'd out lumazine - dt
- Used Prefix and antisense suffix
Ran samples on Gel to confirm sizes
July 10th, 2011
PCR'd out Lumazine Synthase - Dt and RFP
Ran on a 1% agarose gel for 45min at 70volts
July 25th, 2011
Ran 4 samples on an agarose gel:
D22
T22
D24
T24
Preformed a restriction with EcoRI and PstI for Ryan
Ran on 1% agarose gel
July 28th, 2011
Colony PCR of:
k331033-k331035 in pSB1C3
P0440-K331033 in pSB1A2
J04500-LUMAZINE SYNTHASE-DT in pSB1AK3
pBAD-P0440 in pSB1A2
LUMAZINE SYNTASE-DT-pBAD in pSB1A2
July 29th, 2011
Ran PCR samples from July 28th on a 1X TBE Agarose gel
For control comparision of parts from July 28th PCR we ran a PCR of:
K331035
K331033
Lumazine synthase - dt
P0440
Control
EcoRI Restriction of:
J04500-Agn 43 sample 1
J04500 -Agn 43 Sample 2
J04500 - Lumazine synthase - Dt sample 3
K346007
Lumazine synthase - Dt
Ran Restriction on gel to confirm sizes
Notebook team 3 insert picture Page 37
August 2nd, 2011
Transfer of enhanced lumazine synthase from pET-28a into pSB1C3
Restricted with EcoR1 and Pst1
Ran on 1% TAE agarose gel for 90 minutes at 80V
- Gel melted -- unsuccessful.
colonies picked see lab book team 3/ team 1 pg.42
August 4th, 2011
Restriction (
- Enhanced lumazine synthase restricted with EcoR1 and Pst1
- pSB1C3 restricted with EcoR1 and Pst1
- pBAD - P0440 restricted with EcoR1 and Pst1
- lumazine - dt-pBAD -RBS restricted with EcoR1 and Pst1
Gel ran at 80V for 90 minutes
Gel Extraction performed
Ligation
- Enhanced lumazine with pSB1C3
- pBAD - p0440 with pSB1C3
- lumazine - dt -pBAD - RBS with pSB1C3
Transformation
- Transformation of DNA into competent DH5∝ E. coli cells
August 5th, 2011
Transfer of p0440 - K331033 from pSB1A2 into pSB1C3
Restriction
- p0440 - K331033 restricted with EcoR1 and Pst1
Ran on 1% TAE gel for 90 minutes at 80V
August 6th, 2011
Transfer of p0440 - K331033 from pSB1A2 into pSB1C3
Restriction
- p0440 - K331033 restricted with EcoR1 and Pst1
Ran on 1% TAE gel for 90 minutes at 80V
- Gel melted -- unsuccessful
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