Ligations

• Name: Yoyo Y
• 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

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Restriction Digest

• Master Mix
  Milliq	36µl
  BSA	45µl
  10x buffer 2 NEB	45µl

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• 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

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Gel Extraction

• Date: May 4, 2013
• J23108_J61002
• J23113_J61002
• J23114_J61002
• J23100_J61002
• Eluted 50 µL

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Transformation

• 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

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Restriction

• Name: Chris I
• Date: May 7, 2013
• Cut with ‘X’ and ‘P’
• J06702–RBS-mCherry-DT

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Gel Extraction

• Name: Katie T, Elaine B
• Date: May 7, 2013
• J06702
• Eluted at 30µL
• 

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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

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Gel Extraction

• Name: Carissa K
• Date: May 8, 2013
• 2µL of J06702 mix with 4µL of each; J23100, J23108, J23113, J23114 all in plasmid J61002 (amp)
• In the Thermo-cycler at 16°C overnight followed by heat kill.

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PCR of Ligation Mixture(PCR to verify insert in clone vector)

• Name: Chris I
• 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

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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
• 

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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 3µL ligation mix into 30µL of DH5α competent cells, used puc19 control. Heat shocked for 30 seconds at 42°C, added 200µL warm LB, incubated at 37°C for 1 hour. Plated 100µL on amp LB Agar plates. Incubated overnight at 37°C.

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Ligation Round 2 of May 14th Gel Extracted Parts

• 11µL dh20
• 2µL t4 DNA ligase buffer
• 4µL DNA J06702
• 2µL DNA J23100, J23108, J23113, J23114 in plasmid J61002
• 1µL T4 DNA Ligase
• Into the Thermo-cycler overnight at 16°C.

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Picking Cells

• Name: Yoyo Y, Amrinder G, Patrick O, Orion S
• 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

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Picking Cells

• Name: Amrinder G, Orion S
• 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

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10 Fold Miniprep

• Name: Isaac, Patrick
• Date: May 16, 2013
• Spun down 50ml of culture in falcon tubes
• 5000xg twice for 2 mins to collect cells
• Resupended in 1ml solution1 2ml solution 2 and 3, 5ml solution 3
• Spun down at 6000xg for 6 minutes to pellet cell debris
• Eluted into 4 miniprep spin columns twice to reach maximum binding efficiency
• • 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

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Agarose Gel

• 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

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Miniprep

• Name: Amrinder G, Krista F
• 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

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Glycerol Stock

• Name: Amrinder G, Krista F, Yoyo Y, Isaac W
• Date: May 17, 2013
• Glycerol Stocked:
• J23100_J61002
• J23108_J61002
• J23113_J61002
• J23114_J61002

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