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Things we did today

Wetlab work

Checked transformants from yesterday

EtBr stained 2% agarose gel ran at 95V for 1 hour. Ten microliters of each colony PCR reaction were loaded into each well. Positive control was a nir colony PCR (previously confirmed) and negative control was water.

Grace

• Colony PCR'd transformants

• 30 cycles, anneal at 62C, extend for 1 min.
• Ran on EtBr stained 2.0% agarose gel at 95V for 1 hour

• None of the transformations were successful :o(

Determined DNA concentrations of purified RE'd PCR products from 8/4

Grace

Prepared PCR'd nir and J33207 (from yesterday) for transformation/ligation

Grace

• Ran PCR products on EtBr stained 2.0% agarose gel at 60V for 100 min.

• nir band at 330bp confirmed
• J33207 = 4 bands (1.5kb, 2.5kb, 3.2kb, 10kb), none of which are correct

• partsregistry says J33207 DNA is inconsistent

• Extracted nir and 1.5kb J33207 band from gel
• RE digested in 50 μl rxns with EcoRI and SpeI in NEBuffer 2

• Larger rxn volume may improve digest efficiency
• Incubated at 37C for 2.5 hours

• RE digested 10 μl J33207 plasmid prep with EcoRI and SpeI in NEBuffer 2

• To confirm plasmid prep; if good, will use for ligation rxn
• Incubated at 37C for 2 hours

• Ran RE digests on EtBr stained 2.0% agarose gel at 72V for 90 min.

• No visible bands for nir and J33207 RE digest

• We lost a LOT of DNA in the gel purification.

• Band for J33207 plasmid prep RE digest should be ~650bp

• Band at ~850bp = VF2-VR = RE didn't cut???

• Extracted J33207 band (~850) from gel
• Ligated 4.5 μl J33207 with 1.5 μl B0015 (tt)

EtBr stained 2% agarose gel ran at 60V for 100 min. Seven microliters of the PCR reactions were loaded into each lane.

nir (300bp) and (incorrect) J33207 (1.5kb) bands excised from an EtBr stained 2% agarose gel ran at 60V for 100 min.

EtBr stained 2% agarose gel ran at 72V for 100 min. Fifty microliters of the restriction digests were loaded into each well.

J33207 plasmid prep band at ~850bp excised from an EtBr stained 2% agarose gel ran at 72V for 100 min.

Krystle

• Ligated using quick ligase buffer:
  • 3 μl gfpf with 1 μl B0015 (tt)
  • 3 μl gfp with 1 μl B0015 (tt)
  • 3 μl nir with 1 μl B0030 (rbs)
  • 3 μl I14032 with 1 μl B0030 (rbs)

• Used 5 μl ligation reaction to transform 50 μl DB3.1 cells with the the 5 ligations mentioned above

Made LB+amp₁₀₀ plates

Grace and Margaret

Plasmid Prep

Margaret

• pSMC121 was plasmid prepped today

Krystle

• Started 200 ml preps of GFPf, nir, and pRL1383aM

PCR

Margaret

PCR amplification of large quantities of rep, aada, and oriV.

• made large quantities of aadA, rep, and oriV. Please refer to The experiment write-up for more details.

Drylab work

Sequencing

Grace

• Reply from CORE Hawaii

• They will resequence slr1, slr2, BB-pRL1383a for free due to potential mix up of samples and poor quality reads
• For samples that were overloaded when 20ng/100bp sequencing guideline used, try 5 ng/100bp instead.

Discussion

• We're low on small nitrile gloves and Qiagen spin columns. Need to order more. NW
• Be careful when pouring agar plates. Bubbles suck!

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