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Team:Lethbridge Canada/notebook may
From 2013hs.igem.org
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<ul id="notebook_june_links"> | <ul id="notebook_june_links"> | ||
| - | <li class="notebook_link_title"><a href=" | + | <li class="notebook_link_title"><a href="https://2013hs.igem.org/Team:Lethbridge_Canada/notebook_june">June</a></li> |
| - | <li><a href=" | + | <li><a href="https://2013hs.igem.org/Team:Lethbridge_Canada/notebook_june#entry_one">June 1, 2013: Restrictions</a></li> |
| - | <li><a href=" | + | <li><a href="https://2013hs.igem.org/Team:Lethbridge_Canada/notebook_june#entry_two">June 2, 2013: Transformation</a></li> |
| - | <li><a href=" | + | <li><a href="https://2013hs.igem.org/Team:Lethbridge_Canada/notebook_june#entry_three">June 4, 2013: Picked Colonies</a></li> |
| - | <li><a href=" | + | <li><a href="https://2013hs.igem.org/Team:Lethbridge_Canada/notebook_june#entry_four">June 10, 2013: Sent constructs for sequencing</a></li> |
| - | <li><a href=" | + | <li><a href="https://2013hs.igem.org/Team:Lethbridge_Canada/notebook_june#entry_five">Transformation of OXY into BL21</a></li> |
| - | <li><a href=" | + | <li><a href="https://2013hs.igem.org/Team:Lethbridge_Canada/notebook_june#entry_six">Restricted and Ligated and Transformed</a></li> |
| - | <li><a href=" | + | <li><a href="https://2013hs.igem.org/Team:Lethbridge_Canada/notebook_june#entry_seven">Picked Cells</a></li> |
</ul> | </ul> | ||
</div> | </div> | ||
Revision as of 04:02, 17 June 2013
Notebook Links
- March
- March 12, 2013: Transformation
- March 13, 2013: Picking Cell Colonies (Cultures)
- March 13, 2013: Miniprep and Glycerol Stock
- March 15, 2013: Restriction Digestion
- March 16, 2013: Agarose Gel
- March 18, 2013: Agarose Gel
- March 19, 2013: Transformation
- March 20, 2013: Picking Cell Colonies (Cultures)
- March 21, 2013: Miniprep and Glycerol Stock
- March 27, 2013: Transformation
- March 28, 2013: Transformation
- March 28, 2013: Picking Cell Colonies (Cultures)
- March 29, 2013: Picking Cell Colonies (Cultures)
- March 29, 2013: Picking Cells of -80 Glycerol Stock
- April
- April 2, 2013: Restriction - Ligation
- April 2, 2013: Ligation
- April 3, 2013: Transformation
- April 4, 2013: Re-Plating Transformation
- April 5, 2013: Miniprep of ON Cultures
- April 5, 2013: UV Spectroscopy DNA Concentrations
- April 6, 2013: Miniprep From Ligations
- April 7, 2013: PCR
- April 8, 2013: Restriction Digestion of PCR Parts
- April 8, 2013: 3% Agarose Gel of Digested PCR Parts
- April 8, 2013: Gel Extraction
- April 9, 2013: Repeat PCR
- April 9, 2013: Picking Cell Colonies (Cultures)
- April 10, 2013: Miniprep
- April 10, 2013: Restriction
- April 10, 2013: Agarose Gel of PCR 1%
- April 11, 2013: Miniprep
- April 12, 2013: Restriction Digestion
- April 12, 2013: PCR and Cell Reculturization
- April 12, 2013: Miniprep
- April 14, 2013: Miniprep
- April 14, 2013: 1% Agarose Gel
- April 15, 2013: Restriction/Digestion
- April 15, 2013: Restriction/Digestion
- April 15, 2013: 1% Agarose Gel of Promoters
- April 17, 2013: Transformation
- April 30, 2013: 1% Agarose Gel of Promoter Constructs
- May
- May 1, 2013: Ligation
- May 4, 2013: Restriction Digest
- May 4, 2013: 1% Agarose Gel of Promoter Constructs
- May 4, 2013: Gel Extractions
- May 5, 2013: Transformation
- May 7, 2013: Restriction
- May 7, 2013: Gel Extraction
- May 7, 2013: Gel Extracted on Conformation Gel
- May 8, 2013: Ligations
- May 9, 2013: PCR of Ligation Mixture
- May 13, 2013: Restriction of PCR Ligations Gel Conformation
- May 14, 2013: Transformation of Ligations
- May 15, 2013: Ligation
- May 15, 2013: Picking Cells
- May 16, 2013: Picking Cells
- May 16, 2013: 10 Fold Miniprep
- May 17, 2013: Agarose Gel
- May 17, 2013: Miniprep
- May 17, 2013: Glycerol Stock
- May 22, 2013: Ligation
- May 22, 2013: Transformations
- May 23, 2013: Transformations
- May 23, 2013: Picked Oxytocin and Transformations
- May 24, 2013: Glycerol Stock
- May 24, 2013: Miniprep
- May 26, 2013: Miniprep
- May 26, 2013: Restriction Digestion
- May 26, 2013: Agarose gel of Digestion
- May 26, 2013: Digestion Protocol
- May 31, 2013: Agarose Gel
- May 31, 2013: Everything
- May 31, 2013: Everything
Ligations
- Name: Yoyo Yao
- Date: May 1, 2013
- Ligation of Parts – Upstream, Ligated at sites S & P:
- J23100
- J23113
- J23108 +
- J23108 –
- All promoters already attached to Destination Plasmid J61002
- Downstream, Ligated at sites X & P
- J23100
Restriction Digest
- Name: Mackenzie
- Date: May 4, 2013
- 35µL DNA, 14µL MM, 0.5µL Spe1, 0.5µL Pst1
-
Master Mix Milliq 36µl BSA 45µl 10x buffer 2 NEB 45µl
1% Agarose Gel of Promoter Constructs
- Name: Mackenzie
- Date: May 4, 2013
-
Lane # Contents Volume 1 1KB LADDER 6µL 2 J23100_J61002 25µL 3 J23100_J61002 25µL 4 J23100_J61002 25µL 5 J23100_J61002 25µL 6 J23108_J61002 25µL 7 J23108_J61002 25µL 8 J23108_J61002 25µL 9 J23108_J61002 25µL 10 J23113_J61002 25µL 11 J23113_J61002 25µL 12 J23113_J61002 25µL 13 J23113_J61002 25µL 14 J23114_J61002 25µL 15 J23114_J61002 25µL 16 J23114_J61002 25µL 17 J23114_J61002 25µL
Gel Extraction
- Date: May 4, 2013
- J23108 J61002
- J23113 J61002
- J23114 J61002
- J23100 J61002
- Eluted 50 µL
Transformation
- Name: Mackenzie
- Date: May 5, 2013
-
Tube # Part # Plasmid Volume of Competent Cells 1 J23100+J06702 J61002 20µL 2 J23108+J06702 J61002 20µL 3 J23113+J06702 J61002 20µL 4 J23108-J06702 J61002 20µL
Restriction
- Name: Chris Isaac
- Date: May 7, 2013
- Cut with ‘X’ and ‘P’
- J06702 – RBS-mcherry-DT
Gel Extracted Parts on Conformation Gel
- 1µl of 1kb Ladder ng/0.5µg
- 2µl DNA in all samples
| Lane # | Content |
|---|---|
| 1 | Ladder |
| 2 | J06702 |
| 3 | J23113–J61002 mc05/04/13mdy |
| 4 | J23108–J61002 mc05/04/13 |
| 5 | J23100–J61002 mc05/04/13 |
| 6 | J23114–J61002 mc05/04/13 |
| 7 | Ladder |
| 8 | J23100–J 61002 EK150413dmy |
| 9 | J23108–J61002 EK150413 |
| 10 | J23113–J61002EK150413 |
| 11 | J23114 –J61002 EK150413 |
Gel Extraction
- Name: Carissa K.
- Date: May 8, 2013
- 2ul of J06702 mix with 4ul of each; J23100, J23108, J23113, J23114 all in plasmid J61002 (amp)
- In the Thermo-cycler at 16 degrees Celsius overnight followed by heat kill.
PCR of Ligation Mixture(PCR to verify insert in clone vector)
- Name: Chris Isaac
- Date: May 9, 2013
- X7 Master Mix, Taq polymerase
- J23100_J61002 (control)
- J06702_pSB1AK2 (control)
- J23100_J61002_J06702
- J23108_J601002_J06702
- J23113_J61002_J06702
- J23114_J61002_J06702
Restriction of PCR Ligations Gel Conformation
- Name: Patrick O'
- Date: May 13, 2013
- Restriction of May 9th pcr ligation at E and P and ran on 1.0% Gel (refreshed ETH Bromide)
- Ladder lane 2
- 2space
- J06702_pSB1AK2 (control)
- 2spaces
- J23100_J61002 (control)
- spaces
- J23100_J61002_J06702
- J23108_J601002_J06702
- J23113_J61002_J06702
- J23114_J61002_J06702
- Ladder
- Conclusion: Ligations were not Successful
Transformation of Ligations
- Date: May 14, 2013
- Transformed ligation of J06702 with various promoters in plasmid J61002 : J23100, J23108, J23113, J23114 From May 8, 2013.
- Transformed 3ul ligation mix into 30ul of Dh5 alpha competent cells, used puc19 control. Heat shocked for 30second at 42 degrees Celsius added 200ul warm LB, incubated at 37 for 1 hour, Plated 100ul on amp LB Agar plates. Overnight at 37degrees Celsius.
Ligation Round 2 of May 14th Gel Extracted Parts
- 11ul dh20
- 2ul t4 dna ligase buffer
- 4ul DNA J06702
- 2ul DNA J23100, J23108, J23113, J23114 in plasmid J61002
- 1ul T4 dna Ligase
- Into the Thermo-cycler overnight at 16 degrees Celsius.
Picking Cells
- Name: Yoyo, Amrinder, Patrick, Orion
- Date: May 15, 2013
- Put 50µL of Amp resistance into 50mL of Lb media
- Flasks:
-
- J23100_J61002
- J23108_J61002
- J23113_J61002
- J23114_J61002
- J06702_psb1ak2
- Inserted tips of E. Coli into each flask
Picking Cells
- Name: Amrinder, Orion
- Date: May 16, 2013
- Put 5µL of Amp into 5mL of Lb media
- Flasks:
-
- J23100_J61002
- J23108_J61002
- J23113_J61002
- J23114_J61002
- J06702_psb1ak2
- Inserted tips of E. Coli into each tube
10 Fold Miniprep
- Name: Isaac, Patrick
- Date: May 16, 2013
- Spun down 50ml of culture in falcon tubes
- 5000xg twice for 2mins to collect cells
- Resupended in 1ml solution1 2ml solution 2 and 3,5ml solution 3
- Spun down at 6000xg for 6minutes to pellet cell debries
- Eluted into 4 miniprep spin collumns twice to reach maximum binding effeciency
-
- Combined eluted minipreps into one sample totalling 200µL
- Mini prep of (50µL EB):
-
- J23100_J61002
- J23108_J61002
- J23113_J61002
- J23114_J61002
- J06702_PSB1AK2
- Mini-prepped with EZ-10 columns
- Eluted 35µL
- Purified DNA in Erin’s Box
Agarose Gel
- Name: Krista Fjordbotten, Yoyo Yao
- Date: May 17, 2013
- Restriction of:
-
- 4μL DNA
- 1μL Enzyme
- 2.5μL Buffer
- 17.5μL Water
- J23100_J06702 J61002
- J23108_J06702 J61002
- J23113_J06702 J61002
- J23114_J06702 J61002
- J06702 J61002
- Ladder: 2μL Ladder, 0.5μL Dye
- Conformation of all 5 of May 16, 2013 minipreps
-
Well #(from left) DNA Parts Concentration 2 ladder 2μL Ladder/0.5μL Dye 4 J23100_J06702 J61002 25μL DNA/5μL Dye 6 J23108_J06702 J61002 25μL DNA/5μL Dye 8 J23113_J06702 J61002 25μL DNA/5μL Dye 10 J23114_J06702 J61002 25μL DNA/5μL Dye 12 J06702 J61002 25μL DNA/5μL Dye 14 ladder 2μL Ladder/0.5μL Dye
Miniprep
- Name: Amrinder Grewal , Krista Fjordbotten
- Date: May 17, 2013
- Mini prep of (35µL EB):
-
- J23100_J61002
- J23108_J61002
- J23113_J61002
- J23114_J61002
- Mini-prepped with EZ-10 columns
- Eluted 35µL
- Purified DNA in Erin’s Box
- Inserted tips of E. Coli into each flask
Glycerol Stock
- Name: Amrinder Grewal , Krista Fjordbotten, Yoyo Yao, Isaac Ward
- Date: May 17, 2013
- Glycerol Stocked:
- J23100_J61002
- J23108_J61002
- J23113_J61002
- J23114_J61002
