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Revision as of 04:29, 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

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

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, 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 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

[http://manoa.hawaii.edu/ ][http://manoa.hawaii.edu/ovcrge/ ][http://www.ctahr.hawaii.edu ]

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