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Team:Lethbridge Canada/notebook april
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Revision as of 23:56, 6 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
Restriction - Ligation
- Names: Yoyo Y, Wesley M
- Date: April 2, 2013
- Species: E. Coli
- Strain: DH5A
- Protocol: Ute Kothe
- J06702 - mCherry
- J61002 - RFP Reporter
- Promoters: J23113, J23108, J23114, J23100
- Cut Promoters at sites E & S
- Cut mCherry at sites X & P
- Cut Destination Plasmid E1010_pSB1C3 sites E&P
- Promoter Master Mix
- MilliQ: 66.25uL
- BSA: 1.25 uL
- Buffer #2: 12.5 uL
- DNA: 8 uL
- Add 0.5 uL EcoR1 and 0.5 uL Spel to 16 uL MM
- J06702 Master Mix
- MilliQ: 66.25 uL
- BSA: 1.25 uL
- Buffer #2: 12.5 uL
- J06702: 40 uL
- Add 0.5 uL Xbal and 0.5 uL Pst1 to 24MM
- pSB1C3 Master Mix
- MilliQ: 66.25uL
- BSA: 1.25 uL
- Buffer #2: 12.5 uL
- E1010 pSB1C3: 40 uL
- Add 0.5 uL EcR1 and 0.5 uL Pst1 to 24MM
- Incubated at 37 degrees Celsuis for 1 hour
- Heat killed enzyme for 20mins at 65 degrees Celsius
Ligations
- Name: Yoyo Y
- Date: April 2, 2013
- Species: E. Coli
- Strain: DH5A
- Protocol: Ute Kothe
- Tube 1 - J23100_J06702_pSB1C3
- Tube 2 - J23108_J06702_pSB1C3
- Tube 3 - J23113_J06702_pSB1C3
- Tube 4 - J23114_J06702_pSB1C3
- Left overnight at 16 degrees Celsuis
- Agarose Gel of Digested Parts 1%
-
Lane # Contents Volume 1 1 kb DNA Ladder 6µL 2 E1010 pSB1c3 #4 15µL 3 E1010 pSB1c3 #3 15µL 4 E1010 pSB1c3 #2 15µL 5 E1010 pSB1c3 #1 15µL 6 J06702 #4 15µL 7 J06702 #3 15µL 8 J06702 #2 15µL 9 J06702 #1 15µL 10 J23100 15µL 11 J23108 15µL 12 J23113 15µL 13 J23114 15µL
Transformation
- Name: Fiona S, Chris I, Yoyo Y
- Date: April 3, 2013
- Species: E. Coli
- Strain: DH5A
- Protocol: Ute Kothe
- Antibiotic: CAM
- Part Number and Plasmid: J23100_J06702_pSB1C3, J23108_J06702_pSB1C3, J23113_J06702_pSB1C3, J23114_J06702_pSB1C3
- Results: TBA
Re-Plating Transformation
- Names: Chris I, Fiona S
- Date: April 4, 2013
- Species: E. Coli
- Strain: DH5A
- Protocol: Ute Kothe
- Part Number and Plasmid: J23100_J06702_pSB1C3, J23108_J06702_pSB1C3, J23113_J06702_pSB1C3, J23114_J06702_pSB1C3
- Antibiotic CAM
Miniprep of ON Cultures
- Names: Chris I, Yoyo Y
- Date: April 5, 2013
- Species: E. Coli
- Strain: DH5A
- Protocol: Ute Kothe
- Miniprepped J23108_pSB1A2, J23113_pSB1A2, J23114_pSB1A2, J06702_pSB1AK2, used 50uL of Elution Buffer
UV Spectroscopy DNA Concentrations
- Names: Chris I, Yoyo Y, Elaine B
- Date: April 5, 2013
- Species: E. Coli
- Strain: DH5A
- Protocol: Ute Kothe
-
A260 A280 A260/A280 DNA Concentration
at 50-fold
dilution ug/mLJ23108 0.1017 0.0092 11.05435 254.25 J23113 0.1289 0.0338 3.813609 322.25 J23114 0.135 0.0397 3.400504 337.5 J06702-1 0.13 0.0373 3.485255 325
Miniprep From Ligation
- Names: Shikhar M, Krista F, Austin K
- Date: April 6, 2013
- Species: E. Coli
- Strain: DH5A
- Protocol: Ute Kothe
- Miniprepped J23108_J06702_pSB1C3, J23113_J06702_pSB1C3, J23100_J06702_pSB1C3 used 50uL of Elution Buffer
- Note: left plates to grow longer, need DNA sequenced.
PCR
- Name: Erin K
- Date: April 7, 2013
- Species: E. Coli
- Strain: DH5A
- Protocol: Ute Kothe
- J23108_J61002, J23113_J61002, J23114_J61002, J06702_pSb1Ak2
- Separately added 2uL of appropriate DNA and 0.2uL of Pfu Polymerase to 17.8 uL MM
- Agarose Gel confirmation of PCR: Success
-
MM 5x Reagent Volume(uL) MilliQ H2O 67 10X Pfu Buffer with MgSO4 20 10mM dNTPs 2 VF2 5 V4 5
Restriction Digestion of PCR Parts
- Name: Erin K
- April 8, 2013
- Species: E. Coli
- Strain: DH5A
- Protocol: Ute Kothe
- 2 samples of J06702_pSB1AK2: Added 0.5uL XbaI and 0.5uL PstI to 24uL MM
-
J06702 MM 2x Volume (uL) MIlliQ 29.5 BSA 0.5 NEB 10x Buffer 2 5 J06702_pSB1AK2 10 - 2 samples of TAT-E1010_pSB1C3: Added 0.5uL EcoRIand 0.5uL PstI to 24uL MM
-
TAT-E1010_pSB1C3 MM 2x Volume (uL) MIlliQ 12.5 BSA 0.5 NEB 10x Buffer 2 5 TAT-E1010_pSB1C3 30 - 2 samples of each of the 3 promoters: Added 5uL appropriate DNA and 0.5uL EcoRI and 0.5uL SpeI to 16uL MM
-
Promoter MM 7x Volume (uL) MIlliQ 95.75 BSA 1.75 NEB 10x Buffer 2 17.5
3% Agarose Gel of Digested PCR Parts
- Names: Wesley M, Riley M, Krista F
- Date: April 8, 2013
- Species: E. Coli
- Strain: DH5A
- Protocol: Ute Kothe
- Parts: J23108_J61002, J23113_J61002, J23114_J61002, J06702_pSB1ak2
- Results: Unsuccessful
| Lane # | Contents | Volume(µL) |
|---|---|---|
| 1 | Ladder 100bp | 5 |
| 2 | J23108 | 29 |
| 3 | J23108 | 29 |
| 4 | Blank | N/A |
| 5 | J23113 | 29 |
| 6 | Blank | N/A |
| 7 | Blank | N/A |
| 8 | Blank | N/A |
| 9 | J23113 | 29 |
| 10 | Blank | N/A |
| 11 | J23114 | 29 |
| 12 | Blank | N/A |
| 13 | J23114 | 29 |
Gel Extraction
- Names: Wesley M, Riley M, Krista F
- Date: April 8, 2013
- Species: E. Coli
- Strain: DH5A
- Protocol: Biobasic Kit: Eluted with 30uL elution buffer
Repeat PCR
- Name: Elaine B, Chris I, Fiona S, Wesley M, Riley M
- Date: April 9, 2013
- Species: E. Coli
- Strain: DH5A
- Protocol: Ute Kothe
- J23108_J61002, J23113_J61002, J23114_J61002, J06702_pSb1Ak2
- Separately added 2uL of appropriate DNA and 0.2uL of Pfu Polymerase to 17.8 uL MM \
-
MM 5x Reagent Volume (μL) MIlliQ 67 10X Pfu Buffer with MgSO4 2 10mM dNTPs 2 VF2 5 V4 5
Picking Cells
- Names: Chris I, Elaine B, Fiona S, Wesley M, Riley M,
- Date: April 9, 2013
- Species: E. Coli
- Strain: DH5A
- Protocol: Ute Kothe
- Picked: J23113_J61002, J23108_J61002, J23114_J61002, J23100_J61002, J06702_pSB_1AK2
- 5 mL cultures, 5uL Amp. grown overnight at 37 degrees Celsius
Mini Prep
- Names: Yoyo Y
- Date: April 10, 2013
- Species: E. Coli
- Strain: DH5A
- Protocol: Ute Kothe
- Mini Prep of (x2 of 35uL): J06702_pSB1AK3, J23100_J61002, J23108_J61002, J23113_J61002, J23114_J61002
- Mini-prepped with EZ-10 columns
- Eluted 35uL
- Purified DNA in HS iGEM Kit #1
Restriction
- Names: Dylan S
- Date: April 10, 2013
- Species: E. Coli
- Strain: DH5A
- Protocol: Ute Kothe
- Add 33uL DNA to 17uL MM
- 17uL MM to promoter tubes
- 33uL DNA to promoter tubes
- Same procedure for J06702_pSB1AK3
- Incubated at 37 degrees Celsius for 2hours
- Heat killed at 65 degrees Celsius for 20minutes
-
Promoter MM 50μL rxn x4 Volume (μL) MIlliQ 44 BSA 20 NEB 10 x Buffer 2 20
Miniprep
- Names: Dylan S, Patrick O
- Date: April 11, 2013
- Species: E. Coli
- Strain: DH5A
- Protocol: Ute Kothe
- Mini prep of (35uL EB): J06702_pSB1AK3, J23100_J61002, J23108_J61002, J23113_J61002, J23114_J61002
- Mini-prepped with EZ-10 columns
- Eluted 35uL
- Purified DNA in HS iGEM Kit #1
Restriction Digestion
- Names: Wesley M, Yoyo Y
- Date: April 12, 2013
- Species: E. Coli
- Strain: DH5A
- Protocol: Ute Kothe
- Restriction of E1010_pSB1C3 at sites E & P
- 50uL rxn
- Add 30uL DNA to 20uL MM
- Added 0.5uL EcroR1 & Pst1
- Incubated 2hours at 37 degrees Celsius
- Heat killed 20minutes at 65 degrees Celsius
-
MM x 2.5 Volume (μL) MIlliQ 22.5 10x Buffer 2 (NEB) 12.5 BSA 12.5
PCR and Cell Reculturization
- Names: Patrick O, Shikhar M
- Date: April 12, 2013
- Species: E. Coli
- Strain: DH5A
- Protocol: Ute Kothe
- Reculturization of: J06702_pSB1AK3, J23100_J61002, J23108_J61002, J23113_J61002, J23114_J61002
- PCR: Master mix created with: Annealing temperature: 55.6 degrees Celsius
-
MM for Promoters (x5) Conditions for tube "a" MIlliQ 67 μL MIlliQ 13.4 μL 10X Pfu Buffer + MgSO4 20 μL 10X Pfu Buffer + MgSO4 4 μL 10mM dNTPs 2 μL 10mM dNTPs 0.4 μL VF2 5 μL VF2 1 μL VR 5 μL VR 1 μL Pfu polymerase 0.2 μL Pfu polymerase 0.2 μL DNA part 2 μL
Miniprep
- Names: Amrinder G, Steven T
- Date: April 12, 2013
- Species: E. Coli
- Strain: DH5A
- Protocol: Ute Kothe
- Mini prep of (35uL EB): J06702_pSB1AK3, J23100_J61002, J23108_J61002 ,J23113_J61002, J23114_J61002
- Mini-prepped with EZ-10 columns
- Eluted 35uL
- Purified DNA in HS iGEM Kit #1
Miniprep
- Names: Fiona S, Katie T
- Date: April 14, 2013
- Species: E. Coli
- Strain: DH5A
- Protocol: Ute Kothe
- Mini prep of (35uL EB): J23100_J61002, J23108_J61002, J23113_J61002, J23114_J61002, J06702_PSB1AK2
- Mini-prepped with EZ-10 columns
- Eluted 35uL
1% Agarose Gel
- Names: Fiona S, Katie T
- April 14, 2013
- Species: E. Coli
- Strain: DH5A
- Protocol: Ute Kothe
- Parts: J23100_J61002, J23108_J61002, J23113_J61002, J23114_J61002
- Gel Extraction
- Parts: J23108_J61002, J23113 J61002
- Eluted 50 uL
-
Lane # Contents Volume(µL) 1 1 µL Dye, 5µL DNA kB ladder 6 2 J23100_J61002 25 3 J23100_J61002 25 4 J23100_J61002 ~10 5 J23108_J61002 25 6 J23108_J61002 25 7 J23108_J61002 ~10 8 J23113_J61002 25 9 J23113_J61002 25 10 J23113_J61002 ~10 11 J23114_J61002 25 12 J23114_J61002 25 13 J23114_J61002 ~10
Restriction/Digestion
- Master Mix: Yoyo Yao promoters x9
- 35uL DNA, 14uL MM, 0.5uL Spe1, 0.5uL Pst1
- Date: April 15, 2013
-
Master Mix Volume (μL) MIlliQ 36 BSA 45 10x buffer 2 NEB 45
Restriction/Digestion
- Master Mix: FS, KT ½ promoter ½ insulin/glucose x17
- (Promoters) 25uL DNA, 24uL MM, 0.5uL Spe1, 0.5uL Pst1
- (Insulin/Glucose) 25uL DNA, 24uL MM, 0.5uL EcoR1, 0.5uL Pst1
- Date: April 15, 2013
-
Master Mix Volume (μL) MIlliQ 238 BSA 85 10x buffer 2 NEB 85
1% Agarose Gel of Promoters
- Names: Yoyo Y, Erin K
- April 15, 2013
- Species: E. Coli
- Strain: DH5A
- Protocol: Ute Kothe
-
Lane # Contents Volume (μL) 1 1 KB Ladder 6 2 J23100-1 YYSP 25 3 J23100-1 YYSP 25 4 J23100-1 YYSP <10 5 J23100-2 YYSP 25 6 J23100-2 YYSP 25 7 J23100-2 YYSP <10 8 J23108-1 YYSP 25 9 J23108-1 YYSP 25 10 J23108-1 YYSP <10 11 J23108-2 YYSP 25 12 J23108-2 YYSP ~25 -
Lane # Contents Volume (μL) 1 1 KB Ladder 6 2 J23113-1 YYSP 25 3 J23113-1 YYSP 25 4 J23113-2 YYSP 25 5 J23113-2 YYSP 25 6 J23113-2 YYSP <10 7 J23114-1 YYSP 25 8 J23114-1 YYSP 25 9 J23114-2 YYSP 25 10 J23114-2 YYSP 25 -
Lane # Contents Volume (μL) 1 1 KB Ladder 6 2 J23100-1 YYSP 25 3 J23100-1 YYSP 25 4 J23100-1 YYSP 25 5 J23100-2 YYSP 25 6 J23100-2 YYSP <25 7 J23108-1 YYSP 25 8 J23108-1 YYSP 25 9 J23108-1 YYSP 10 10 J23108-2 YYSP 25 11 J23108-2 YYSP 25 -
Lane # Contents Volume (μL) 1 1 KB Ladder 6 2 J23113-1 YYSP 25 3 J23113-1 YYSP 25 4 J23113-2 YYSP 25 5 J23113-2 YYSP 25 6 J23114-1 YYSP <10 7 J23114-1 YYSP 25 8 J23224-1 YYSP 25 9 J23114-2 YYSP 10 10 J23114-2 YYSP 25
Transformation
- Name: Dylan S
- Date: April 17, 2013
- Species: E. Coli
- Strain: DH5A
- Protocol: Ute Kothe
-
Tube # Part # Plasmid Volume of Competent Cells(µL) 1 J23100+J06702 J61002 100 2 J23108+J06702 J61002 100 3 J23113+J06702 J61002 50 4 J23114+J06702 J61002 50 5 J23100 mlc J06702 PSB3C5 50 6 J23100 mlc J06702 PSB3C5 50 +/- Controls PUC19 Amp resistance 50 each
1% Agarose Gel of Promoter Constructs
- Name: Chris I
- Date: April 30, 2013
- Species: E. Coli
- Strain: DH5A
- Protocol: Ute Kothe
-
Lane # Contents Volume(µL) 1 1 KB Ladder 6 2 J23100_J61002 25 3 J23100_J61002 25 4 J23100_J61002 25 5 J23100_J61002 25 6 J23108_J61002 25 7 J23108_J61002 25 8 J23108_J61002 25 9 J23108_J61002 25 10 J23108_J61002 <20 11 J23108_J61002 25 13 1 KB Ladder 25 12 J23100_J61002 <20 1 1 KB Ladder 6 2 J23113_J61002 25 3 J23113_J61002 25 4 J23113_J61002 <20 5 J6702_pSB1AK2 20 6 J6702_pSB1AK2 20 7 J6702_pSB1AK2 20 8 J6702_pSB1AK2 25 9 J6702_pSB1AK2 25 10 J6702_pSB1AK2 20 11 J6702_pSB1AK2 20 12 J6702_pSB1AK2 20 13 None N/A
