Team:Brown-Stanford/Lab/Notebook/Week6

July 18, 2011

PowerCell

• imaged PCR: RBSm|cscB on 7/15, according to 7/11 parameters (may need to remove from thermocycler):
• first gel from 7/18: unable to see ladder or bands (restained with fresh GelStar, then recast a fresh gel to try loading again)
• did not use TAE for post-stain; HAVE TO USE TAE + GELSTAR FOR POST STAIN OR ELSE DNA WILL DIFFUSE OUT OF GEL
• gel extract and/or PCR cleanup to prepare for Gibson
• imaged PCR: second round Ana wk/med/str, Nostoc wk (from 7/15)
• Gel extracted and PCR cleanup directly on rbs-GFP-TTsp (bands cut 7/15, PCR product from 7/12); Nanodrop results: PCR cleanup-120ng/ul, Gel Extract- 19ng/ul
• possibly run a 2nd round PCR? (don’t think so)
• PCR (put in at 6:30pm):
• 2nd round PCR on RBSw cscB, RBSs cscB, cscB TTsp from 7/15 gel exts
• redid second round PCR on Ana wk/med/str, Nostoc wk, RBSm|cscB, (+) control 15B
• Jovian and Lei tested sucrose assay

FRETSensor

• Finally got the constructs ordered (3 from Invitrogen, and the cohesin-cohesin from DNA 2.0)

July 19, 2011

PowerCell

• imaged PCR from 7/18 (9 samples); MAKE SURE TO STAIN WITH 5ul GelStar in 50ml TAE
• gel bands visible/cut/extracted for RBSw cscB (~10ng/ul), RBSm cscB (~5ng/ul), RBSs cscB (13.9ng/ul),
• Jovian extracted bands for Ana med, Nos weak: Nanodrop readings in the negatives? REDO
• processed ATCC E coli 9637 into liquid culture
• resuspended dried cells into 450ul LB, put 150ul of suspension with 2ml of LB, placed in 37C at 3pm (repeated 3x)
• set up an 85ml BG11 N+ culture for Anabaena 7120 and N. punctiforme (75ml BG11N+ and 10ml of the existing saturated liquid culture)

REGOBricks

• Consolidated protocols for transformations, including:
  • Soil
  • Protoplast
  • Protoplast electroporation
  • Standard

July 20, 2011

PowerCell

• PCRed: the never-successful ones: cscB|RBSgfp; Nostoc strong RBS; Ana weak/str (did not see bands)
• made 3x cryostocks of E. coli W (slots 47, 48, 49 in PowerCell -80C box); used 20% glycerol
• imaged the PCR from this morning (7/20):
• under 2% agarose, poststaining for 30min is inadequate; Kosuke says to use fresh GelStar in polyethylene container, stain for more like 45min
• INSTEAD, begin to cast gels with GelStar embedded, use 6ul of ladder
• second gel (Gel #1b) cast, loaded with all 15ul of remaining PCR product; 6ul of ladder
• Anabaena strong showed strong bands, bands cut, ready for extraction
• Lei transformed 2x cscB plasmid into TOP10
• transformed 100ul TOP10 cells with 2ul of hydrated CscB; x2 replicates; 25min on ice, 2min heat shock @ 42C, then add 500ul LB and place in 37C for ~20min
• two plates in 37C at 5:30pm
• Sigma order was placed with Mike (b-NAD purchased, 600mg)

FRETSensor

sick! :(

July 20, 2011

REGOBricks

• Began preparing for the arrival of pUB110
  • Made solutions for 4 transformation protocols
  • Autoclaved potting soil in preparation for soil transformation

• • Performed OD curve with 1x, 1/2x, 1/5x, 1/10x, and 1/100x with Bang media and cuvettes. Instead of plating, used haemocytometer to count cells in all samples.Came up with a final cell count equation, Cell count/mL = (OD - .0746)x10E9

7-23

• Began dried filter paper experiment:
• [Sterile in UV] Took nitrocellulose paper**, put into on large plastic petri dishes
• 12 sheets of paper, divided into 2 x [control, 10x dilution, 100x dilution, 1000x dilution and 10000x dilution] of S. pasteurii (OD600: 1._____)
• prewet nitrocellulose paper with 100 ul of DI water, tip plate around to spread it out
• put 50 ul of each cell dilution onto each paper, spread evenly
• incubate paper in giant petri dish in 30 C

• Controls II: repeat same procedure, but instead of incubating the paper, immediately flip the paper (cell containing side down) onto Bang media plates. Incubate plates at 30 C

• Grew up
  • 2x 2mL B. subtilis in LB media
  • 2x 2mL E. coli in LB media + amp
    • caution, nitrocellulose paper is extremely flammable

July 21, 2011

PowerCell

• gel extraction on Ana strong from 7/20 PCR; Nanodrop 7.2ng/ul
• imaged Nostoc strong (from 7/20 PCR); moderately strong bands cut, extracted (Nanodrop 5.5ng/ul)
• made 1L BG11 N+ from 50X stock
• PCR: Phusion PCR
• tried gradient PCR 40-50C using phusion polymerase; samples Ana mid, Nos mid, RBSm|cscB, pos cont (well 15B)
• used 1.5ul of template in 20ul rxn; added 3% DMSO
• times: initial colony popping with 98C for 5’; initial denature 30sec @ 98C; 10sec @ 98; 20sec @ 40-50C; 15sec @ 72C; repeat 32x; final elongation 10min @ 72C
• Phusion is FAST, yo
• pick colony of CscB transformant, start 2ml LB amp+ liquid culture (three replicates); placed in 37C at 7:15pm

July 22, 2011

PowerCell

• Imaged Phusion PCR from 7/22; 4x bright visible bands cut from all samples (Ana med, Nos med, RBSm|cscB)
• gel extraction on bands (ProMega kit; re-eluted samples through column three times on first and last steps in order to capture maximum DNA)
• Nanodrop readings: RBSm|cscB 40.1ng/ul; Ana med 21.1ng/ul; Nos med 14.2 ng/ul
• Started more 100ml cultures of Anabaena and Nostoc; BG11 N+, 2x of each
• Plasmid prep of liquid cscB cultures
• plasmids in 4C of Rm335, “Norman’s Plasmids” rack; Nanodrop readings of 100.6, 79.4, 92.2ng/ul
• Dry autoclave run for tips and glassware (with Biocementation team)
• embed bacteria on nitrocellulose paper for monday balloon flight