Team:Hawaii/Notebook/2008-08-30

= Things we did today =

Construction of p+r and re-replacement of BB-pRL1383a MCS

 * Grace


 * Ran RE digests from last night on agarose gel
 * C0012: correct vector band ~2.1kb
 * J33207: band at ~700bp (only one enzyme cut -- XbaI did not cut; PstI cuts because C0012 was cut by PstI) and ~850bp (uncut PCR product)
 * nir: ~800bp band (from GFP contaminant in plasmid prep, cut only once) and ~2kb band (??)
 * BB-pRL1383a: ran WAY too much product. Will redo RE digest with 20% of plasmid used. Since J33207 was only cut once, it's assumed BB-pRL1383a was cut only once as well.
 * Extracted C0012 vector band from gel
 * Ligated:
 * plac + rbs (B0034)
 * PCR of J33207
 * RE digested:
 * p+r with EcoRI, XbaI, SpeI, PstI
 * J33207 and BB-pRL1383a with EcoRI and PstI
 * Treated C0012 vector (pSB1A2) with SAP

Construction of Broad-host-Range plasmid

 * Margaret


 * PCR:
 * rep(50ul rxn), pRL1383a (template), rep(primers)
 * P1 lytic(50ul rxn), pSB2K3(extracted from BB paper), P1(primers)
 * (+) control: Base Vector + VF2&VR, (-) control: water + VF2&VR
 * 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

PCR

 * Margaret
 * P1 lytic (50ul rxn, pSB2K3(extracted from BB paper), P1(primers), 0.1ug/ml BSA
 * omega (25ul rxn) The last gel had a ladder (so does this reaction, see gel from tomorrow).

Added parts to registry

 * Margaret


 * plac+B0030:
 * plac+B0034:
 * oriT: K125320
 * P1 lytic: K125330
 * oriV:K125340
 * rep proteins:K125800
 * rep+oriV

= Discussion =

= Quote of the Day = "History is the only laboratory we have in which to test the consequences of thought. - Étienne Gilson"