Team:Hawaii/Notebook/2008-08-11

= Things we did today =

Checked plasmid prep from weekend

 * Grace


 * Ran on 2.0% agarose gel to verify plasmids
 * DNA didn't run. Agarose concentration too high. Redid on 0.8% gel.
 * Genomic DNA up top?
 * Clean prep (no RNA)!
 * Only E0240 verified. All other bands wrong size (circular/supercoiled?). Need RE digest to verify.
 * Checked DNA concentrations via nanodrop spectrometer

Made 1000x Amp100 stock solution

 * Grace

Reinoculated for cryostocking

 * Grace


 * I14032 from 2005 and 2008 distributions

Construction of GFP device

 * Grace


 * Extracted nir+rbs, plac+rbs, GFP, GFPf from gel ran yesterday
 * B0015 could not be extracted because fragment was not visible under short wave UV
 * Digestion was done for 3A assembly rather than rear ligation (oops). Redid RE digest.
 * Checked DNA concentrations via nanodrop spectrometer


 * Restriction digested in 30 &mu;l reactions, incubated at 37C for 2 hours:
 * B0015 with XbaI then EcoRI
 * GFP and GFPf with EcoRI and SpeI
 * slr1, slr2, pilA with SpeI and PstI
 * Ran new RE digests EtBr stained 2% agarose gel at 72V for 1.5 hours
 * Extracted parts from gel and determined DNA concentrations


 * Ligated for 1 hour using Quick T4 DNA Ligase and Quick Ligase buffer:
 * 8 &mu;l GFP + 0.5 &mu;l B0015
 * 4 &mu;l GFPf + 4 &mu;l B0015
 * 2 &mu;l GFPf + 1.5 &mu;l slr1
 * 2 &mu;l GFPf + 3.5 &mu;l pilA
 * Transformed 7 &mu;l ligation reaction into DB3.1 cells
 * RE digest overnight of 22 &mu;l pSB1A2 with EcoRI and PstI for 3A assembly

Testing restriction enzymes in the lab's -20C freezer

 * Grace


 * Digested pRL1383a with BamHI (should result in a single linear fragment)
 * Digested pRL1383a with HindIII (should result in a single linear fragment)
 * Digested plasmid preps (E0240, I14032, I51020, nir+rbs, plac+rbs) with NotI (should result in two fragments -- vector and insert)

Ligation of pRL1383a Parts

 * Margaret


 * restriction digest of rep, oriV, aada(BB), aada(pRL1383a), P1 lytic region, pSB1A3, B0030, B0015
 * Ligation: rep+B0030, oriV+pSB1A3, aadA(BB)+B0030, aadA(pRL1383a)+B0030, P1 lytic + B0015, pSB1A3 to itself (-) control
 * Transformation into DH5-a (batch 3)

Started Culture for plasmid prep & cryostocks

 * to be completed 8/12
 * B0015, pSB3K3, oriT(cryostock & plasmid prep), B0030, I14032, E0040, J33207

= Discussion =
 * FYI:
 * According to the Endy lab, ligation reactions should have <100ng DNA per reaction for maximum efficiency
 * ~10ng vector should be used in ligation reactions (6:1 ratio of insert to vector)

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