Team:Hawaii/Notebook/2008-08-11

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:<strong>Grace</strong>
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Revision as of 03:55, 12 August 2008

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Things we did today

Wetlab work

Checked plasmid prep from weekend

Grace
EtBr stained 0.8% agarose gel ran at 95V for 1 hour. Five microliters of plasmid were loaded into each well.
  • 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
Plasmid DNA concentration 260/280 260/230
E0240 757.7 ng/μl 2.06 1.49
I14032 (2005 distribution) 541.4 ng/μl 2.01 1.27
I51020 2775.6 ng/μl 1.97 1.77
nir+rbs 566.8 ng/μl 1.83 1.10
plac+rbs 344.0 ng/μl 1.95 1.28

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
Part DNA concentration
nir+rbs 4.8 ng/μl
plac+rbs 3.6 ng/μl
GFP 4.7 ng/μl
GFPfusion 6.4 ng/μl
EtBr stained 2% agarose gel ran at 72V for 1.5 hours. Thirty microliters of the RE digest reactions were loaded into each well.
  • Restriction digested in 30 μl reactions:
  • 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
Part DNA concentration
slr1 2.6 ng/μl
pilA 1.1 ng/μl
GFP 0.4 ng/μl
GFPf 11.3 ng/μl
B0015 1.9 ng/μl
  • Ligated for 1 hour using Quick T4 DNA Ligase and Quick Ligase buffer:
  • 8 μl GFP + 0.5 μl B0015
  • 4 μl GFPf + 4 μl B0015
  • 2 μl GFPf + 1.5 μl slr1
  • 2 μl GFPf + 3.5 μl pilA
  • Transformed 7 μl ligation reaction into DB3.1 cells
  • RE digest overnight of 22 μl 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

Restriction digest after 2 hours.
Ligation reaction after 2 hours.
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


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