Team:WashU/Notebook

From 2011.igem.org

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**Add 1.25mL glycerol to yeast culture
**Add 1.25mL glycerol to yeast culture
**Freeze yeast culture in -80 degree freezer
**Freeze yeast culture in -80 degree freezer
 +
 +
*Make E.coli mini-preps
 +
**Transfer to E.coli culture tubes
 +
**Centrifuge for one minute at 12000g
 +
**Pour out supernatant
 +
**add 100uL of resuspension solution with RNase
 +
**add 200uL of lysis buffer
 +
**add 350uL of neutralization buffer
 +
**Centrifuge for 10 minutes at 12000g

Revision as of 18:19, 4 June 2011


This is a template page. READ THESE INSTRUCTIONS.
You are provided with this team page template with which to start the iGEM season. You may choose to personalize it to fit your team but keep the same "look." Or you may choose to take your team wiki to a different level and design your own wiki. You can find some examples HERE.
You MUST have a team description page, a project abstract, a complete project description, a lab notebook, and a safety page. PLEASE keep all of your pages within your teams namespace.


You can write a background of your team here. Give us a background of your team, the members, etc. Or tell us more about something of your choosing.
WashU logo.png

Tell us more about your project. Give us background. Use this is the abstract of your project. Be descriptive but concise (1-2 paragraphs)

File:WashU team.png
Your team picture
Team Example


Home Team Official Team Profile Project Parts Submitted to the Registry Modeling Notebook Safety Attributions


Contents

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.

May 31

  • Preliminary Shopping List
    1. PCR buffer w/o MgCl - 5ML - $45.00
    2. M8787- 5ML -MgCl Reagent- $49.30
    3. D7295-.5ML - dNTP - $73.00
    4. D4184-250UN - Taq Polymerase - $150.00
    5. D8045-250UN - AccuTaq - $300
    6. E1385-5ML – Ethidium Bromide – $23.70
    7. T8280-1L – Tris Acetate EDTA Buffer – $45.10
    8. NA1020-1KT – PCR Clean-Up Kit -$102.50
    9. NA1111-1KT - Gel Extraction Kit - $102.00

Subtotal: $890.60

  • Met with Cohen Lab grad students to finalize plan and get cassettes.
  • Received Leu2 cassette in pRS305 plasmid.
  • Received Ura3 cassette in pRS306 plasmid.

June 1

  • Lesson from Bert Berla on how to design primers
  • Codon Optimized the four genes using tool at: http://www.encorbio.com/protocols/Codon.htm
    • Double checked gene and restriction sites to make sure enzyme does not cut in the middle of the gene
  • Design Primers for cassettes
    • Ura3, Leu2, KanMX4, and NatMX4

June 2

  • Made two liters of LB solution
    • 25g of LB Broth per Liter (10g Tryptone, 5g Yeast Extract, and 10g NaCl)
    • Autoclaved solutions: 30 minute sterilization.
    • Refrigerated after cooling.

June 3

  • Made YPD
    1. In 800ml water in 1 L bottle dissolve 10g of BactoYeast extract
    2. Dissolve 20g of BactoPeptone in the above solution
    3. In 200ml water in 500ml bottle dissolve 20g Dextrose
    4. Autoclave both, combine after autoclaving. (30 minute sterilization)
  • Made 1000X Ampicillin Stock
    • Added 2g Ampicillin to 20ml water.
    • Added NaOH until dissolved.
    • Sterile filtered.
    • Frozen in -20C Freezer
  • Made cultures of E. coli strains with KanMx4 and Natmx4 inserts.
    • In culture tubes, added 5ml LB and 5ul 1000X Ampicillin Stock.
    • Transferred one colony per strain from plates to culture tubes.
    • Incubate at 30 degrees Celsius shaking at 225rpm overnight.
  • Made cultures of Yeast strains BC177 and BC178
    • Added 5ml YPD to two culture tubes.
    • Transferred one colony per strain from plates to culture tubes.
    • Incubate at 30 degrees Celsius shaking at 225rpm overnight.

June 4

  • Making freezer stocks of yeast culture
    • Add 1.25mL glycerol to yeast culture
    • Freeze yeast culture in -80 degree freezer
  • Make E.coli mini-preps
    • Transfer to E.coli culture tubes
    • Centrifuge for one minute at 12000g
    • Pour out supernatant
    • add 100uL of resuspension solution with RNase
    • add 200uL of lysis buffer
    • add 350uL of neutralization buffer
    • Centrifuge for 10 minutes at 12000g
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