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	<ul>		<ul>
	<li>Quantification of digestion EXSP products</li>		<li>Quantification of digestion EXSP products</li>
-	<li>Replate RBS+lasR</li>	+	</ul>
-	<li>Ligation of PCR qteE in pGEM vector</li>	+	<li>Replate RBS+lasR</li>
-	<li>Transformation:</li>	+	<li>Ligation of PCR qteE in pGEM vector</li>
-	<ul>	+	<li>Transformation:</li>
-	<li>BBa_J04450</li>	+	<ul>
-	</ul>	+	<li>BBa_J04450</li>
-	<li>Digestion</li>	+	</ul>
-	<ul>	+	<li>Digestion</li>
-	<li>BBa_B0015</li>	+	<ul>
-	</ul>	+	<li>BBa_B0015</li>
	</ul>		</ul>
	</ul>		</ul>

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Revision as of 04:14, 24 September 2014

WELCOME TO iGEM 2014! Your team has been approved and you are ready to start the iGEM season! On this page you can document your project, introduce your team members, document your progress and share your iGEM experience with the rest of the world! Click here to edit this page!
Home Team Official Team Profile Project Parts Modeling Notebook Safety Attributions
Notebook
You should make use of the calendar feature on the wiki and start a lab notebook. This may be looked at by the judges to see how your work progressed throughout the summer. It is a very useful organizational tool as well.

Lab Protocols

Competent Cell with Calcium Chloride

1. Materials

• Solid LB medium - 1 plate
• Liquid LB medium
• 2 250ml centrifuge tubes
• Centrifuge
• Cell sample (DH5-alpha in our case)
• Autoclave
• Inoculation loop
• Liquid nitrogen
• 37°C oven
• CaCl₂:
• 60mM CaCl₂
• 10mM HEPES
• 15% glicerol
• H₂O to 100ml

2. Methods

Day 1

1. Risk the LB plate with the cell sample (DH5-alpha)
2. Incubate the plate at 37°C overnight

Day 2

1. Sterilize in the Autoclave
• 100ml of liquid LB medium
• 100 ml of CaCl₂
• 2 250ml centrifuge tubes
2. Prepare a 6ml LB inoculum with a DH5-alpha colony and incubate at 37°C/250rpm overnight

Day 3

1. Add 2ml of the inoculum in 100ml of liquid LB medium. Incubate at 37°C/250rpm until the OD reaches 0,375
2. Distribute the volume in 2 250ml centrifuge tubes (pre chilled) and spin at 10000rpm/7min/4°C
3. Discard the supernatant and ressuspend the pellet in 10ml of CaCl₂ solution
4. Spin the tubes at 7000rpm/7min/4°C
5. Discard the supernatant and ressuspend the pellet in 10ml of CaCl₂ solution. Incubate 30min on ice
6. Spin at 7000rpm/7min/4°C
7. Discard the supernatant and ressuspend the pellet in 2ml of the CaCl₂ solution
8. Distribute the 2ml in 500ul tubes adding 50ul in each.
9. Freeze the samples in liquid nitrogen
10. Stock at -80°C

Transformation in Escherichia coli (DH5-alpha)

1. Materials

• 1,5ml tube
• Styrofoam box with ice
• Agar plate with antibiotic
• Competent cells
• Plasmidial DNA
• Centrifuge
• Water bath at 42°C
• Liquid LB medium
• Shaker

2. Methods

1. Briefly spin the competent cells and put then on ice
2. Add 50ng of plasmidial DNA in a 1,5ml tube
3. Add the 50ul of competent cell in the same tube
4. Keep the tube on ice for 25min
5. Put the tube in a 42° water bath for 2 min
6. Put the tube on ice for 5 min
7. Add 200ul of liquid LB
8. Incubate at 250rpm/37°C/1 hour
9. Plate the the solution in a Agar plate with the appropriate antibiotic
10. Incubate the plate at 37°C overnight

Assemblies

• Assembly Form
• AI
• AII
• AIII
• AV
• AVI
• BII
• BIII
CII
• DI
• KI
• KIII
• KIV
• KVI
• KVIII
• KX
• KXI

Life Inside the LAB

July

Monday	Tuesday	Wednesday	Thursday	Friday	Saturday	Sunday
30/06 PCR lasR (BBa_C0079) for amplification + RBS addition using primers	01/07 Purification of lasR PCR product	02/07 Transformation of the Biobricks: BBa_143012 BBa_K081001 BBa_E0840 Inoculum of the Biobricks sent by iGEM HQ: BBa_K316037 BBa_K316018 BBa_K316015	03/07 Transformation of the Biobricks sent by the Imperial College Team: BBa_K316016 BBa_K143031 Miniprep, Quantification of the plasmidial DNA samples and Restriction Analysis: BBa_143012 BBa_K081001 BBa_E0840	04/07	05/07	06/07 Inoculum: BBa_143012 BBa_K081001 BBa_E0840
07/07 Miniprep, Quantification and Restriction Analysis: BBa_143012 BBa_K081001 BBa_E0840 Transformation: BBa_B0015	08/07	09/07 Inoculum: BBa_B0015	10/07 Miniprep, Quantification and Restriction Analysis: BBa_B0015 (restriction analysis fail, repeat) PCR qteE for amplification + RBS addition using primers Ligation of RBS+lasR in PTZ57R/T vector (instaclone kit)	11/07 Prepare 40% glycerol solution Transformation: RBS + lasR (BBa_C0079) Purification: RBS + qteE	12/07	13/07
14/07 Assembly:AIII Digestion EXSP Prepare X-gal Prepare more LB medium (solid + liquid)	15/07 Assembly:AIII Quantification of digestion EXSP products Replate RBS+lasR Ligation of PCR qteE in pGEM vector Transformation: BBa_J04450 Digestion BBa_B0015	16/07	17/07	18/07	19/07	20/07
21/07	22/07	23/07	24/07	25/07	26/07	27/07
28/07	29/07	30/07	31/07

August

Monday	Tuesday	Wednesday	Thursday	Friday	Saturday	Sunday
	30/07	01/08	02/08	03/08	Points	Points
First Name	Last Name	Points	Points	Points	Points	Points
First Name	Last Name	Points	Points	Points	Points	Points
First Name	Last Name	Points	Points	Points	Points	Points
First Name	Last Name	Points	Points	Points	Points	Points

September

Monday	Tuesday	Wednesday	Thursday	Friday	Saturday	Sunday
	30/07	01/08	02/08	03/08	Points	Points
First Name	Last Name	Points	Points	Points	Points	Points
First Name	Last Name	Points	Points	Points	Points	Points
First Name	Last Name	Points	Points	Points	Points	Points
First Name	Last Name	Points	Points	Points	Points	Points

October

Monday	Tuesday	Wednesday	Thursday	Friday	Saturday	Sunday
	30/07	01/08	02/08	03/08	Points	Points
First Name	Last Name	Points	Points	Points	Points	Points
First Name	Last Name	Points	Points	Points	Points	Points
First Name	Last Name	Points	Points	Points	Points	Points
First Name	Last Name	Points	Points	Points	Points	Points

Retrieved from "http://2014.igem.org/Team:Brasil-SP/Notebook"