Team:Brown-Stanford/Lab/Notebook/Week7

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== ''' July 24, 2011''' ==
 +
===REGOBricks===
 +
*Control II of yesterday's experiment grew colonies in 10x and 100x dilution, colonies located at edges of nitro paper
 +
 +
*Attempted revival of ? of yesterday’s filter paper experiment:
 +
**flip the paper upside down (side containing cells facing down) onto new Bang plates. The moisture of the plate covers the paper.
 +
**On one half of the paper, add 50 ul Bang media, to wet/wash the cells onto the plate.
 +
 +
*Repeat dried filter paper experiment with Subtilis (OD 0.996) and E. coli (OD 1.12) grown up from yesterday.
 +
 +
*Transformation of E. coli (competent Top10) with D1 plasmid containing amp resistance
 +
 +
*Growing up 50 mL of E. coli containing cscB plasmid in LB + amp
 +
 +
*Made BANG plates with a gradient of Kan to test for S. pasteurii survival
 +
*Data will be used to make plates to select for Kan resistance in transformed pasteurii
 +
== ''' July 25, 2011''' ==
 +
===PowerCell===
 +
*Perform additional Phusion PCRs:
 +
*Round 1: cscB|TTsp; cscB|RBSgfp; rbsGFPttsp; (no pos control)
 +
*cscB|TTsp bands visualized, cut; the rest were inconclusive; redo
 +
*cscB|RBSgfp was distorted on gel; both CG and GFP exhibited lots of secondary amplifications; try higher specific
 +
*make BG11 N+ plates (in prep for NDA) (see NDA3 design document)
 +
*Test transformation - liquid cultures of conjugative transformation E. coli lines
 +
*pRL1383a - from plate
 +
*pRL443 - from plate
 +
*RP1 - from cryo
 +
*pRL25 - from plate
 +
*pDS4101 - from agar
 +
*make liquid culture of E. coli W for NDA3 - from cryo
 +
*Innoculated 5 more 500mL liquid cultures for both Ana and Nos
 +
*Tranformed J45120 and J45200 for long (+) conts. for PCRs
 +
== ''' July 26, 2011''' ==
 +
===PowerCell==
 +
*gel extraction on cscB|TTsp from 7/25/11
 +
*PCR cscB|RBSgfp; rbsGFPttsp (WITH PROPER TEMPLATE); Ana str, Nos str, RBSs|cscB; gradient from 55-45C
 +
*design primer for prom-GFP-ttsp construct;
 +
*Prepare test transformation with prepared liquid cultures (eek)
 +
*make three classes of plates (BG11 agar + 5% LB; BG11 agar + spec; BG11 agar + kan)
 +
*BG11 + LB agar plates (more like ~160ml BG11, 16.6ml LB, ~180ml agar); 133ml BG11 + ~133ml agar + 335ul spec100; 133ml BG11 + ~133ml agar + 335ul kan20
 +
*See cyanobacterial transformation document for protocol
 +
*Liquid cultures of transformation plasmid strains, again.
 +
*transformed j45200 long pos cont.
 +
*gel extracted cscb|TTSP phusion PCR. 32.9 ng\ul
 +
*miniprep long pos controls
 +
*PCR m.AvaI??????????
 +
== ''' July 27, 2011''' ==
 +
===PowerCell===
 +
*Image PCR from 7/26 (rbsGFPttsp; cscB|rbsGFP; Ana str; Nos str; RBSsCscB);
 +
*samples are still in the gradient cycler in Innovation Lab (turn off after removing tubes!);
 +
*run the 20 lanes from 7/26 (five templates x four temperatures), re-run the four lanes of Nostoc med part (from 7/21 PCR) in order to obtain more gel extracted product
 +
*Gel 1 (0.7%): 1kb ladder, rbsGFPttsp; cscB|rbsGFP
 +
*Gel 2 (2%): 100bp ladder, Ana str, Nos str
 +
*Gel 3 (2%): 100bp ladder, RBSsCscB, Nos med
 +
*Try to doublecheck GelRed procedure with Thomas before making gels
 +
*Extract any good bands (follow Thomas’ suggested modifications to maximize yield), Nanodrop readings
 +
*Another stab at transformation!
 +
*made BG11+1% Agar+5% LB, BG11+1% Agar+Spec, BG11+1%Agar+Kan plates
 +
*Continued transformation begun 7/26 with Julius’ plates, incubating for one day in 30?
 +
*Began new transformation on new plates, incubating in 30?
 +
*Setup cross of pDS4101 and pRL25 to incubate overnight, to be streaked tomorrow on amp/kan LB.
 +
*Liquid cultures involved in conjugative transformation are stored in 335 4?.
 +
*Ran Gels on Julius’ PCR from 26Jy2011 (Cscb|RBSgfp, RBS-GFP-TTSP, RBSs|cscB, Ana S, Nos S) cut out bands.
 +
*Lots of extraneous amplification, especially in cscB|RBSgfp - cut out bands in right places, but  sort of dubious they’re the right things...
 +
*Innoculated 4mL liquid stock of long pos cont. for mini prep.
 +
== ''' July 28, 2011''' ==
 +
===PowerCell===
 +
*Streak pDS4101xpRL25 onto amp/kan (lei/jovian)
 +
*gel extraction of 1st round PCR’s
 +
*miniprep of long positive control from registry
 +
*2nd round PCR’s
 +
*Gel of 2nd round PCR’s
 +
*Gel extraction of 2nd round PCR’s
 +
*continue transformation-post-conjugation cells streaked onto antibiotic BG11 for attempts 1 and 2
 +
*lab cleaning at 1:30
 +
===REGOBricks===
 +
*Successful balloon launch this morning (Max tell us more!)
 +
*Plasmid prepped pUB110 using the modified qiagen protocol w/ lysozyme.
 +
*Ran a gel to confirm the presence of the plasmid which is ~4500 bp
 +
*Today was lab clean up day. Yipee
 +
== ''' July 29, 2011''' ==
 +
===PowerCell===
 +
*ran gel of 2nd round PCRs
 +
*cut out bands, gel purified
 +
*successfully got
 +
*PCR amplification of pSB1C3 and pSB1A3 with g1001, g0001 primers
 +
*Create and aliquot Gibson master mix

Revision as of 20:26, 25 September 2011

Brown-Stanford
iGEM

July 24, 2011

REGOBricks

  • Control II of yesterday's experiment grew colonies in 10x and 100x dilution, colonies located at edges of nitro paper
  • Attempted revival of ? of yesterday’s filter paper experiment:
    • flip the paper upside down (side containing cells facing down) onto new Bang plates. The moisture of the plate covers the paper.
    • On one half of the paper, add 50 ul Bang media, to wet/wash the cells onto the plate.
  • Repeat dried filter paper experiment with Subtilis (OD 0.996) and E. coli (OD 1.12) grown up from yesterday.
  • Transformation of E. coli (competent Top10) with D1 plasmid containing amp resistance
  • Growing up 50 mL of E. coli containing cscB plasmid in LB + amp
  • Made BANG plates with a gradient of Kan to test for S. pasteurii survival
  • Data will be used to make plates to select for Kan resistance in transformed pasteurii

July 25, 2011

PowerCell

  • Perform additional Phusion PCRs:
  • Round 1: cscB|TTsp; cscB|RBSgfp; rbsGFPttsp; (no pos control)
  • cscB|TTsp bands visualized, cut; the rest were inconclusive; redo
  • cscB|RBSgfp was distorted on gel; both CG and GFP exhibited lots of secondary amplifications; try higher specific
  • make BG11 N+ plates (in prep for NDA) (see NDA3 design document)
  • Test transformation - liquid cultures of conjugative transformation E. coli lines
  • pRL1383a - from plate
  • pRL443 - from plate
  • RP1 - from cryo
  • pRL25 - from plate
  • pDS4101 - from agar
  • make liquid culture of E. coli W for NDA3 - from cryo
  • Innoculated 5 more 500mL liquid cultures for both Ana and Nos
  • Tranformed J45120 and J45200 for long (+) conts. for PCRs

July 26, 2011

=PowerCell

  • gel extraction on cscB|TTsp from 7/25/11
  • PCR cscB|RBSgfp; rbsGFPttsp (WITH PROPER TEMPLATE); Ana str, Nos str, RBSs|cscB; gradient from 55-45C
  • design primer for prom-GFP-ttsp construct;
  • Prepare test transformation with prepared liquid cultures (eek)
  • make three classes of plates (BG11 agar + 5% LB; BG11 agar + spec; BG11 agar + kan)
  • BG11 + LB agar plates (more like ~160ml BG11, 16.6ml LB, ~180ml agar); 133ml BG11 + ~133ml agar + 335ul spec100; 133ml BG11 + ~133ml agar + 335ul kan20
  • See cyanobacterial transformation document for protocol
  • Liquid cultures of transformation plasmid strains, again.
  • transformed j45200 long pos cont.
  • gel extracted cscb|TTSP phusion PCR. 32.9 ng\ul
  • miniprep long pos controls
  • PCR m.AvaI??????????

July 27, 2011

PowerCell

  • Image PCR from 7/26 (rbsGFPttsp; cscB|rbsGFP; Ana str; Nos str; RBSsCscB);
  • samples are still in the gradient cycler in Innovation Lab (turn off after removing tubes!);
  • run the 20 lanes from 7/26 (five templates x four temperatures), re-run the four lanes of Nostoc med part (from 7/21 PCR) in order to obtain more gel extracted product
  • Gel 1 (0.7%): 1kb ladder, rbsGFPttsp; cscB|rbsGFP
  • Gel 2 (2%): 100bp ladder, Ana str, Nos str
  • Gel 3 (2%): 100bp ladder, RBSsCscB, Nos med
  • Try to doublecheck GelRed procedure with Thomas before making gels
  • Extract any good bands (follow Thomas’ suggested modifications to maximize yield), Nanodrop readings
  • Another stab at transformation!
  • made BG11+1% Agar+5% LB, BG11+1% Agar+Spec, BG11+1%Agar+Kan plates
  • Continued transformation begun 7/26 with Julius’ plates, incubating for one day in 30?
  • Began new transformation on new plates, incubating in 30?
  • Setup cross of pDS4101 and pRL25 to incubate overnight, to be streaked tomorrow on amp/kan LB.
  • Liquid cultures involved in conjugative transformation are stored in 335 4?.
  • Ran Gels on Julius’ PCR from 26Jy2011 (Cscb|RBSgfp, RBS-GFP-TTSP, RBSs|cscB, Ana S, Nos S) cut out bands.
  • Lots of extraneous amplification, especially in cscB|RBSgfp - cut out bands in right places, but sort of dubious they’re the right things...
  • Innoculated 4mL liquid stock of long pos cont. for mini prep.

July 28, 2011

PowerCell

  • Streak pDS4101xpRL25 onto amp/kan (lei/jovian)
  • gel extraction of 1st round PCR’s
  • miniprep of long positive control from registry
  • 2nd round PCR’s
  • Gel of 2nd round PCR’s
  • Gel extraction of 2nd round PCR’s
  • continue transformation-post-conjugation cells streaked onto antibiotic BG11 for attempts 1 and 2
  • lab cleaning at 1:30

REGOBricks

  • Successful balloon launch this morning (Max tell us more!)
  • Plasmid prepped pUB110 using the modified qiagen protocol w/ lysozyme.
  • Ran a gel to confirm the presence of the plasmid which is ~4500 bp
  • Today was lab clean up day. Yipee

July 29, 2011

PowerCell

  • ran gel of 2nd round PCRs
  • cut out bands, gel purified
  • successfully got
  • PCR amplification of pSB1C3 and pSB1A3 with g1001, g0001 primers
  • Create and aliquot Gibson master mix


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