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Things we did today

Wetlab work

Construction of p+r (cont.) and re-replacement of BB-pRL1833a MCS

EtBr stained 0.8% agarose gel ran at 60V for 1.5 hours.
  • Ran RE digests from last night on gel
  • plac+rbs band not visible; will ligate using plac and rbs parts (instead of plac+rbs part)
  • Image to right has been edited w/ Photoshop. Faint smear for plac+rbs was not visible before.
  • Extracted bands from gel
  • Ligated:
  • plac and rbs (B0034) and C0012 vector
  • J33207 and BB-pRL1383a
  • Transformed into DH5α cells
  • Plated BB-pRL1383a+J33207 on selective media with and without IPTG to verify if IPTG is necessary for blue/white screening in this strain

Inoculated TB+sp100 with BB-pRL1383a


Construction of Broad-Host-Range Plasmid Parts

File:Re digest 8 31 08.jpg
Restriction digest of rep and P1 lytic regions.
  • gel of yesterday's re-digest, lane 3 was cleaned using gel purification spin columns and stored in -20°C. Lanes 7-10 were extracted and cleaned as well.
  • Ligation: using T4 ligase, 10X buffer (from the same lot as the enzyme, it was aliquoted today). 2 hour incubation at room temperature. The reaction was placed in 4°C in case transformation does not yield colonies.
insert vector
1 pSB1A3 cut with X,P(de-P042-)18ng 3 rep (PCR product) X,P 82ng
1pSB1A3 cut with X,P(de-P042-)35.6ng 3P1 lytic region X,P 64.4ng
3pSB1A3 cut with X,P(de-P042-)66.2ng 1 rep (PCR product) X,P 33.8ng\
  • Transformation: of ligation products from today into DH5-alpha cells. Everything plated on Amp100 LB plates, pSMC121 used as positive control (plated on SmSp).
  • Plasmid Prep: stored in -20°C in 30ul TE buffer
  • Culture: 5mL Terrific Broth + amp100
  • plac+B0030 (1:6):3, 4, 7
  • plac+B0030 :3
  • plac+B0034 (1:6):1
  • plac+B0034 (1:3):3


Quote of the Day

History is the only laboratory we have in which to test the consequences of thought. - √Čtienne Gilson