Team:Penn/Notebook/Biofilms

From 2012.igem.org

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

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