Team:Stanford/Notebook/Lab Work/Week 1

From 2010.igem.org

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===Alex's Notebook===
===Alex's Notebook===
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Transformations using DH5alpha cells from Graham (also have TOP10)
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*Transformations using DH5alpha cells from Graham (also have TOP10)
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Protocol:
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*Protocol:
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1) Take out competent E.coli cells from –80 C freezer.
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**1) Take out competent E.coli cells from –80 C freezer.
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2) Turn on water bath to 42 C.
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**2) Turn on water bath to 42 C.
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3) Put 50 ul of competent cells in a 1.5 ml tube (Eppendorf or similar).
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**3) Put 50 ul of competent cells in a 1.5 ml tube (Eppendorf or similar).
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4) KEEP EVERYTHING ON ICE UNLESS OTHERWISE STATED.
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**4) KEEP EVERYTHING ON ICE UNLESS OTHERWISE STATED.
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5) Add 50 ng or 2.5/5 ul of circular DNA into E.coli cells. DO NOT PIPETTE UP OR DOWN; STIR INSTEAD! Incubate on ice for 15/30 min.
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**5) Add 50 ng or 2.5/5 ul of circular DNA into E.coli cells. DO NOT PIPETTE UP OR DOWN; STIR INSTEAD! Incubate on ice for 15/30 min.
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6) Put tube(s) with DNA and E.coli into water bath at 42 C for 20 seconds.
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**6) Put tube(s) with DNA and E.coli into water bath at 42 C for 20 seconds.
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7) Put tubes back on ice for 15 minutes to reduce damage to the E.coli cells.
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**7) Put tubes back on ice for 15 minutes to reduce damage to the E.coli cells.
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8) Add 1 ml of SOC. Incubate tubes for 1/2 hour(s) at 37 C.
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**8) Add 1 ml of SOC. Incubate tubes for 1/2 hour(s) at 37 C.
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9) Spread about 100/200 ul of the resulting culture on LB plates (with the appropriate antibiotic added – usually Ampicillin or Kanamycin). Grow overnight.
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**9) Spread about 100/200 ul of the resulting culture on LB plates (with the appropriate antibiotic added – usually Ampicillin or Kanamycin). Grow overnight.
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10) Pick the colonies about 12-16 hours later.
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**10) Pick the colonies about 12-16 hours later.
Parts:
Parts:

Revision as of 23:19, 4 August 2010

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Contents

6/28 Monday

General Meeting Notes

  • Wiki stuff
    • Everyone should sign up on 2010.igem.org website
      • Take advantage of the new calendar system
      • Need to transfer current website over
    • Current wiki needs a new vision
      • Francisco + Alex assembling wiki committee
  • Transform the first wave of cells
    • Pick colonies
    • Colony PCR
    • Sequence parts to validate
      • Use the Smolke sequencing network
  • Characterize promoters
    • Digest, ligate
    • Need to acquire enzymes
    • Need an PO account.
      • Email Jeanne
  • Get competent cells
  • Getting DNA
    • Assembly PCR
    • PCR from host cells
    • Synthesize de novo
  • RNA team
    • Use RNAstructure
      • Maybe use ViennaRNA or dinamelt
    • Talk to Arkin and Berkeley Postdocs
    • Finalize sRNA library
    • 9kb plasmid
  • Meeting scheudle
    • Monday morning is good for logistics
    • Group meetings for presentations
      • One person presents their work in the context of everything else
    • Journal club
    • Karina will set up a doodle


Alex's Notebook

  • Transformations using DH5alpha cells from Graham (also have TOP10)
  • Protocol:
    • 1) Take out competent E.coli cells from –80 C freezer.
    • 2) Turn on water bath to 42 C.
    • 3) Put 50 ul of competent cells in a 1.5 ml tube (Eppendorf or similar).
    • 4) KEEP EVERYTHING ON ICE UNLESS OTHERWISE STATED.
    • 5) Add 50 ng or 2.5/5 ul of circular DNA into E.coli cells. DO NOT PIPETTE UP OR DOWN; STIR INSTEAD! Incubate on ice for 15/30 min.
    • 6) Put tube(s) with DNA and E.coli into water bath at 42 C for 20 seconds.
    • 7) Put tubes back on ice for 15 minutes to reduce damage to the E.coli cells.
    • 8) Add 1 ml of SOC. Incubate tubes for 1/2 hour(s) at 37 C.
    • 9) Spread about 100/200 ul of the resulting culture on LB plates (with the appropriate antibiotic added – usually Ampicillin or Kanamycin). Grow overnight.
    • 10) Pick the colonies about 12-16 hours later.

Parts:  Plasmids: - 1A2  P1, 11P, BBa_J04450. 100-300, pMB1, 2079. - 1A3  P1, 1C, Ba_J04450. 100-300, pMB1, 2157. - 4A5  P1, 1G, BBa_J04450. ~5, pSC101, 3395. - 3C5  P1, 3C, BBa_J04450. 10-12, p15A, 2738. - 2K3  P1, 5C, Insert: BBa_J04450, star. Length: 4425 bp.

 Promoters: - Bad (BBa_I0500). Input(s): L-arabinose. Output(s): PoPS. Need with mutated strain. Tested range: 2e-4% up to 2e-1%. Can probably go lower. (http://mic.sgmjournals.org/cgi/reprint/147/12/3241).  P1, 14N, Plasmid: pSB2K3. Length: 1210 bp. - TetR and LuxR (BBa_F2620). Input(s): C10HSL. Output(s): PoPS. Tested range: ~1e-7 up to 1e-4. Can probably go to a higher concentration. (http://partsregistry.org/wiki/images/f/fe/EndyFig1-F2620DataSheet.pdf)  P2, 6E, Plasmid: pSB1A2, star. Length: 1061 bp.

 Reporter: - Endy’s GFP (BBa_E0240).  P1, 12M Plasmid: pSB1A2, star. Length: 876 bp.

 Miscellaneous Parts: - GFP Producer Controlled by 3OC6HSL Receiver Device (BBa_T9002). For use with C10HSL (homoserine lactone; http://www.sigmaaldrich.com/catalog/ProductDetail.do?lang=en&N4=17248|SIGMA&N5=SEARCH_CONCAT_PNO|BRAND_KEY&F=SPEC)  P2, 9A, Plasmid: pSB1A3, star. Length: 1945 bp.

6/29 Tuesday

Laura's Notebook

  • made LB agar (with Alex and Chris)
    • made 1 500 mL bottle, 3 250 mL bottles, then autoclaved

6/30 Wednesday

Laura's Notebook

  • Re-try failed transformation (failed 2X for Alex and Chris)
  • Protocol followed:
  1. get competent cells from -80oC freezer
  2. set H2O bath to 42oC (already done)
  3. add 50 uL cells to a 1.5 mL tube (keep tubes on ice)
  4. add 50 ng or 2.5 uL circular DNA to E. Coli (variation: used 5.0 uL I0500 this time)
  5. on ice 30 min
  6. 24oC H2O bath 20 sec.
  7. on ice 15 min
  8. add 1 mL SOC, 2 hours at 37oC (rotating)
  9. plate100uL on appropriate plate (kanamycin this time), incubate 12-16 hours before picking colonies
  • variations: 200 uL plated this time, also plated on kan/X-gal/IPTG plate (plasmid inducible copy # by X-gal)

7/1 Thursday

Laura's Notebook

  • did 8 minipreps from Chris'/Alex's cultures
  • used Qiagen spin kit, with the following variations:
  1. skipped "recommended" wash step
  2. eluted with H2O
  • Concentrations from Nanodrop (taken by Chris B.)
psb1A2: 58 ng/uL
psb1A3: 53 ng/uL
psb2K3: 40 ng/uL
psb4A5: 63.4 ng/uL
E0240: 65 ng/uL
T9002: 104 ng/uL
F2620: 57.5ng/uL
  • helped Karina, Francisco with the transformations of parts

Francisco's Notebook

7/2 Friday

Recap Meeting Notes

  • Things to think about:
    • name of the device (see if electronic analogue exists)
    • decouple the small RNA binding from the target gene ("RSID" tags)
    • leadership structure for the team

Francisco's Notebook

  • Miniprepped Parts. Nanodrop results below:
Part 260/280 260/230 ng/uL
B1006 1.85 1.62 84.0
B0015 1.87 1.84 40.3
B0034 1.91 1.82 52.2
E0040 1.88 1.94 85.4
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