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Things we did today

Wetlab work

Restriction Digest

Grace

• Sequential digest of B0015 with XbaI and EcoRI

Checked transformants from yesterday's ligations

Grace

PCR

Grace & Krystle

Colony PCR. EtBr stained 2.5% agarose gel. Two microliters of the PCR reactions were loaded into each well.

PCR extractionof Biobricks. EtBr stained 2.5% agarose gel ran at 60V for 2 hours. Five microliters of each reaction were loaded into each well.

• Colony PCR'd all transformants from yesterday's ligations and slr1, slr2, nir, pilA, GFPf

• Annealed at 58C, elongated for 90 sec.

• PCR of E0240 and I14032 from filter paper

• Annealed at 58C, elongated for 90 sec.

• Ran on 2.5% gel

• Contaminant at 250bp
• pRL1383a = 5 bands (~1.2kb, ~0.9kb, ~0.35kb, ~0.3kb, ~0.25kb); desired band = 958bp
• nir+rbs = 2 bands (~0.275kb, ~0.25kb); desired band = 353bp

• nir was not inserted
• E/X sites not compatible; ligase ligated blunt ends created by RE digest leftovers?

• GFPf+tt = 2 bands (~0.32kb, ~0.25kb); desired band = 1081bp

• GFPf not inserted

• Extracted E0240, slr1, slr2, GFP, pilA, I14032 from gel (correct sizes)

• GFPf = ~1150bp (too big, what's going on? we're consistently getting GFPf this big)
• nir = ~1100bp (too big)
• Since sequencing returned correct sequences for GFPf and nir, regrow E. coli from colony used for sequencing and see if desired/expected results can be obtained or if results are the same

Inoculated LB+amp₁₀₀

Grace

• GFP
• nir

Made LB media

Krystle

Discussion

Quote of the Day

Logically, it makes sense, but we're not always very logical people so it's still a weird numbering system - KS, GK in reference to the BioBrick part numbers

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