Team:Brown-Stanford/Lab/Notebook/Week6

From 2011.igem.org

(Difference between revisions)
(Created page with "{{:Team:Brown-Stanford/Templates/Main}} <html> <div id="notebook"> </html> <html> </div> </html> {{:Team:Brown-Stanford/Templates/Foot}}")
Line 5: Line 5:
</html>
</html>
 +
== ''' 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
 +
== ''' 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)
 +
== ''' 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

Revision as of 20:22, 25 September 2011

Brown-Stanford
iGEM

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

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)

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


Retrieved from "http://2011.igem.org/Team:Brown-Stanford/Lab/Notebook/Week6"