Team:Penn/Notebook/Biofilms

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Revision as of 19:28, 17 July 2012 by Qiaop (Talk | contribs)

Contents

6/6/12

  • Set up equipment
  • Autoclaved 2x 1L Sterile ddH2O
  • Autoclaved 500 mL LB Broth for scale up of luxS (Plate 3 Well 2H, pSB1A2), plsr (Plate 2 Well 14H in pSB1A2 resistance to Amp)
  • Added iGEM logo to wiki

6/7/12

  • Resuspended available biobricks luxS & plsr (resuspended in 10uL ddH2O)
  • Resuspended DNA transformed into DH5α (40uL DH5α+2uL DNA) & plated on LB plates w/ 15 uL of 100 mg/mL Amp spread on surface
  • Requested lsrR & lsrK from iGEM HQ

6/8/12

  • iGEM Wiki now split into two separate Notebooks
  • Plenty of colonies, too many to count(TMTC) grew from plsr & luxS transformation from 6/7/12
    • Selected one colony each
    • Grew in 10 mL LB+Amp (100 ug/mL)
  • Contacted researchers for V. harveyi bioassay & lysostaphin, V. harveyi must be purchased through ATCC ($300), lysostaphin obtained thru MTA.

6/9/12

  • ~2mL of LB evaporated during incubation
  • Cells spun down @ 5000 rpm for 10 min
  • Miniprepped 4x Lysis into 1 column
  • [plsr]=112 ng/uL
  • [luxS]=114 ng/uL

6/11/12-6/16/12

  • Dry lab work

6/18/12

  • Obtained & resuspended primers for eGFP & plsr

6/19/12

  • performed PCR w/ NEB standard taq kit w/ primers for eGFP & plsr
  • Tested 2 annealing temperatures, 55C and 50C
  • Ran gel w/ PCR product (2% agarose), saw clear eGFP bands, but possible .1kb obscured by loading dye.

6/20/12

  • Performed qiagen PCR purification & nanodropped (it like it's hot)

6/21/12

  • Ran gel of yesterday's PCR purification
SCIENCE.
Image of 2% Agarose Gel of post-purification PCR products of eGFP (.85kb) & plsr (.1kb)
    • EtBr Gel 2% Agarose
      • Lane 1: NEB 2-log ladder (1 ug loaded)
      • Lane 2: PCR of eGFP using plsr_XhoI Fwd & eGFP_EcoRI Rev primers w/ 55C annealing temp.
      • Lane 3: PCR of plsr using plsr_BglII Fwd & plsr_XhoI Rev primers w/ 55C annealing temp.
      • Lane 4: PCR of eGFP using plsr_XhoI Fwd & eGFP_EcoRI Rev primers w/ 50C annealing temp.
      • Lane 5: PCR of plsr using plsr_BglII Fwd & plsr_XhoI Rev primers w/ 50C annealing temp.
      • Lane6/7: Null
      • Lane 8: 1kb ladder left at rt o/n
  • Obtained & transformed pET-26b

6/22/12

  • Miniprepped pet26b and digested with BglII and EcorR

6/25/12

  • Ran gel of eGFP/plsr ligation
  • Ordered restriction enzymes from the Cell Center

6/26/12

  • Attempted Gel Purification of digest products from 2/25/12
    • Yields for Gel purification were too low to be useful (>1 ng/uL)
  • Digested purified PCR rxns (annealing temperature=55C)

6/27/12

Ran gel of digested PCR rxns (55C), found no evidence of successful digestion.

6/28/12

Purified digest and attempted

6/29/12

  • Performed digest of pET-26b, eGFP, and plsr
  • Spread Biobrick shipment on Amp plates (lsrR, lsrK)

7/2/12

  • Selected colonies from lsrK and lsrR streaked plates
    • Grew in 10mL TB Broth

7/3/12

  • Ran gel of ligated product, no size shift, unlikely that ligation was successful.
  • Ligation rxn transformed to make sure

7/5/12

  • Ligation transformation of pET-26-plsr-GFP failed, no colonies present
  • Troubleshooting of pET-26 digestion
    • Digest of pET-26 w/ BglII and XhoI
    • Time course:
    • 10 min
    • 30 min
    • 3 hr
    • 6 hr
    • o/n

7/6/12

  • Redesigned primers for plsr & egfp