From 2008.igem.org

Birds Eye  View Team	Projects	Events	Resources
Sponsors	Experiments	Milestones	Protocols
	Notebook (t)	Meetings (t)

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

• Recent changes
• What links here
• Related changes
• Special pages
• My preferences

• Printable version
• Permanent link
• Privacy policy
• Disclaimers