From 2013hs.igem.org

(Difference between revisions)

Revision as of 19:58, 19 June 2013

Restriction - Ligation

Names: Yoyo Y, Wesley M
Date: April 2, 2013
Species: E. coli
Strain: DH5α
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: 66.25µL
- BSA: 1.25µL
- Buffer #2: 12.5µL
- DNA: 8µL
- Add 0.5µL EcoR1 and 0.5µL Spel to 16µL MM
J06702 Master Mix
- MilliQ: 66.25µL
- BSA: 1.25µL
- Buffer #2: 12.5µL
- J06702: 40µL
- Add 0.5µL Xbal and 0.5µL Pst1 to 24MM
pSB1C3 Master Mix
- MilliQ: 66.25µL
- BSA: 1.25µL
- Buffer #2: 12.5µL
- E1010 pSB1C3: 40µL
- Add 0.5µL EcR1 and 0.5µL Pst1 to 24MM
Incubated at 37°C for 1 hour
Heat killed enzyme for 20 mins at 65°C

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Ligations

Name: Yoyo Y
Date: April 2, 2013
Species: E. coli
Strain: DH5α
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°C
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

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Transformation

Name: Fiona S, Chris I, Yoyo Y
Date: April 3, 2013
Species: E. coli
Strain: DH5α
Protocol: Ute Kothe
Antibiotic: CAM
Part Number and Plasmid: J23100_J06702_pSB1C3, J23108_J06702_pSB1C3, J23113_J06702_pSB1C3, J23114_J06702_pSB1C3
Results: TBA

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Re-Plating Transformation

Names: Chris I, Fiona S
Date: April 4, 2013
Species: E. coli
Strain: DH5α
Protocol: Ute Kothe
Part Number and Plasmid: J23100_J06702_pSB1C3, J23108_J06702_pSB1C3, J23113_J06702_pSB1C3, J23114_J06702_pSB1C3
Antibiotic CAM

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Miniprep of ON Cultures

Names: Chris I, Yoyo Y
Date: April 5, 2013
Species: E. coli
Strain: DH5α
Protocol: Ute Kothe
Miniprepped J23108_pSB1A2, J23113_pSB1A2, J23114_pSB1A2, J06702_pSB1AK2, used 50µL of Elution Buffer

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UV Spectroscopy DNA Concentrations

Names: Chris I, Yoyo Y, Elaine B
Date: April 5, 2013
Species: E. coli
Strain: DH5α
Protocol: Ute Kothe

	A260	A280	A260/A280	DNA Concentration at 50-fold dilution ug/mL
J23108	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

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Miniprep From Ligation

Names: Shikhar M, Krista F, Austin K
Date: April 6, 2013
Species: E. coli
Strain: DH5α
Protocol: Ute Kothe
Miniprepped J23108_J06702_pSB1C3, J23113_J06702_pSB1C3, J23100_J06702_pSB1C3 used 50µL of Elution Buffer
Note: left plates to grow longer, need DNA sequenced.

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PCR

Name: Erin K
Date: April 7, 2013
Species: E. coli
Strain: DH5α
Protocol: Ute Kothe
J23108_J61002, J23113_J61002, J23114_J61002, J06702_pSB1AK2
Separately added 2µL of appropriate DNA and 0.2µL of Pfu Polymerase to 17.8µL MM
Agarose Gel confirmation of PCR: Success

MM 5x Reagent	Volume(µL)
MilliQ H2O	67
10X Pfu Buffer with MgSO4	20
10mM dNTPs	2
VF2	5
V4	5

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Restriction Digestion of PCR Parts

Name: Erin K
April 8, 2013
Species: E. coli
Strain: DH5α
Protocol: Ute Kothe
2 samples of J06702_pSB1AK2: Added 0.5µL XbaI and 0.5µL PstI to 24µL MM

J06702 MM 2x	Volume (µL)
MIlliQ	29.5
BSA	0.5
NEB 10x Buffer 2	5
J06702_pSB1AK2	10

2 samples of TAT-E1010_pSB1C3: Added 0.5µL EcoRIand 0.5µL PstI to 24µL MM

TAT-E1010_pSB1C3 MM 2x	Volume (µL)
MIlliQ	12.5
BSA	0.5
NEB 10x Buffer 2	5
TAT-E1010_pSB1C3	30

2 samples of each of the 3 promoters: Added 5µL appropriate DNA and 0.5µL EcoRI and 0.5µL SpeI to 16µL MM

Promoter MM 7x	Volume (µL)
MIlliQ	95.75
BSA	1.75
NEB 10x Buffer 2	17.5

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3% Agarose Gel of Digested PCR Parts

Names: Wesley M, Riley M, Krista F
Date: April 8, 2013
Species: E. coli
Strain: DH5α
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

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Gel Extraction

Names: Wesley M, Riley M, Krista F
Date: April 8, 2013
Species: E. coli
Strain: DH5α
Protocol: Biobasic Kit: Eluted with 30µL elution buffer

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Repeat PCR

Name: Elaine B, Chris I, Fiona S, Wesley M, Riley M
Date: April 9, 2013
Species: E. coli
Strain: DH5α
Protocol: Ute Kothe
J23108_J61002, J23113_J61002, J23114_J61002, J06702_pSB1AK2
Separately added 2µL of appropriate DNA and 0.2µL of Pfu Polymerase to 17.8µL MM

MM 5x Reagent	Volume (μL)
MIlliQ	67
10X Pfu Buffer with MgSO4	2
10mM dNTPs	2
VF2	5
V4	5

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Picking Cells

Names: Chris I, Elaine B, Fiona S, Wesley M, Riley M,
Date: April 9, 2013
Species: E. coli
Strain: DH5α
Protocol: Ute Kothe
Picked: J23113_J61002, J23108_J61002, J23114_J61002, J23100_J61002, J06702_pSB_1AK2
5mL cultures, 5µL Amp. grown overnight at 37°C

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Mini Prep

Names: Yoyo Y
Date: April 10, 2013
Species: E. coli
Strain: DH5α
Protocol: Ute Kothe
Mini Prep of (x2 of 35µL): J06702_pSB1AK3, J23100_J61002, J23108_J61002, J23113_J61002, J23114_J61002
Mini-prepped with EZ-10 columns
Eluted 35µL
Purified DNA in HS iGEM Kit #1

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Restriction

Names: Dylan S
Date: April 10, 2013
Species: E. coli
Strain: DH5α
Protocol: Ute Kothe
Add 33µL DNA to 17µL MM
17µL MM to promoter tubes
33µL DNA to promoter tubes
Same procedure for J06702_pSB1AK3
Incubated at 37°C for 2 hours
Heat killed at 65°C for 20 minutes

Promoter MM 50μL rxn x4	Volume (μL)
MIlliQ	44
BSA	20
NEB 10 x Buffer 2	20

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Agarose Gel of PCR 1%

Result: Successful

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Miniprep

Names: Dylan S, Patrick O
Date: April 11, 2013
Species: E. coli
Strain: DH5α
Protocol: Ute Kothe
Mini prep of (35µL EB): J06702_pSB1AK3, J23100_J61002, J23108_J61002, J23113_J61002, J23114_J61002
Mini-prepped with EZ-10 columns
Eluted 35µL
Purified DNA in HS iGEM Kit #1

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Restriction Digestion

Names: Wesley M, Yoyo Y
Date: April 12, 2013
Species: E. coli
Strain: DH5α
Protocol: Ute Kothe
Restriction of E1010_pSB1C3 at sites E & P
50µL rxn
Add 30µL DNA to 20µL MM
Added 0.5µL EcoR1 & Pst1
Incubated 2 hours at 37°C
Heat killed 20 minutes at 65°C

MM x 2.5	Volume (μL)
MIlliQ	22.5
10x Buffer 2 (NEB)	12.5
BSA	12.5

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PCR and Cell Reculturization

Names: Patrick O, Shikhar M
Date: April 12, 2013
Species: E. coli
Strain: DH5α
Protocol: Ute Kothe
Reculturization of: J06702_pSB1AK3, J23100_J61002, J23108_J61002, J23113_J61002, J23114_J61002
PCR: Master mix created with: Annealing temperature: 55.6°C

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

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Miniprep

Names: Amrinder G, Steven T
Date: April 12, 2013
Species: E. coli
Strain: DH5α
Protocol: Ute Kothe
Mini prep of (35µL EB): J06702_pSB1AK3, J23100_J61002, J23108_J61002 ,J23113_J61002, J23114_J61002
Mini-prepped with EZ-10 columns
Eluted 35µL
Purified DNA in HS iGEM Kit #1

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Miniprep

Names: Fiona S, Katie T
Date: April 14, 2013
Species: E. coli
Strain: DH5α
Protocol: Ute Kothe
Mini prep of (35µL EB): J23100_J61002, J23108_J61002, J23113_J61002, J23114_J61002, J06702_pSB1AK2
Mini-prepped with EZ-10 columns
Eluted 35µL

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1% Agarose Gel

Names: Fiona S, Katie T
April 14, 2013
Species: E. coli
Strain: DH5α
Protocol: Ute Kothe
Parts: J23100_J61002, J23108_J61002, J23113_J61002, J23114_J61002
Gel Extraction
Parts: J23108_J61002, J23113_J61002
Eluted 50 µL

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

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Restriction/Digestion

Master Mix: Yoyo Yao promoters x9
35uL DNA, 14µL MM, 0.5µL Spe1, 0.5µL Pst1
Date: April 15, 2013

Master Mix	Volume (μL)
MIlliQ	36
BSA	45
10x buffer 2 NEB	45

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Restriction/Digestion

Master Mix: FS, KT ½ promoter ½ insulin/glucose x17
(Promoters) 25µL DNA, 24µL MM, 0.5µL Spe1, 0.5µL Pst1
(Insulin/Glucose) 25µL DNA, 24µL MM, 0.5µL EcoR1, 0.5µL Pst1
Date: April 15, 2013

Master Mix	Volume (μL)
MIlliQ	238
BSA	85
10x buffer 2 NEB	85

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1% Agarose Gel of Promoters

Names: Yoyo Y, Erin K
April 15, 2013
Species: E. coli
Strain: DH5α
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

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Transformation

Name: Dylan S
Date: April 17, 2013
Species: E. coli
Strain: DH5α
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

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1% Agarose Gel of Promoter Constructs

Name: Chris I
Date: April 30, 2013
Species: E. coli
Strain: DH5α
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

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 						<li><a href="https://2013hs.igem.org/Team:Lethbridge_Canada/notebook_march#entry_fourteen">March 29, 2013: Picking Cells of -80 Glycerol Stock</a></li>
 					</ul>
+<img src="https://static.igem.org/mediawiki/2013hs/e/ea/Lethbridge_hs_igem_2013_banner_notbookapril.png">
 					<ul id="notebook_april_links">

Team:Lethbridge Canada/notebook april

From 2013hs.igem.org

Revision as of 19:58, 19 June 2013

Restriction - Ligation

Ligations

Transformation

Re-Plating Transformation

Miniprep of ON Cultures

UV Spectroscopy DNA Concentrations

Miniprep From Ligation

PCR

Restriction Digestion of PCR Parts

3% Agarose Gel of Digested PCR Parts

Gel Extraction

Repeat PCR

Picking Cells

Mini Prep

Restriction

Agarose Gel of PCR 1%

Miniprep

Restriction Digestion

PCR and Cell Reculturization

Miniprep

Miniprep

1% Agarose Gel

Restriction/Digestion

Restriction/Digestion

1% Agarose Gel of Promoters

Transformation

1% Agarose Gel of Promoter Constructs