Team:Hawaii/Notebook/2008-08- 6

= Things we did today =

Checked transformants from yesterday

 * 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 &mu;l 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 &mu;l 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 &mu;l J33207 with 1.5 &mu;l B0015 (tt)


 * Krystle
 * Ligated using quick ligase buffer:
 * 3 &mu;l gfpf with 1 &mu;l B0015 (tt)
 * 3 &mu;l gfp with 1 &mu;l B0015 (tt)
 * 3 &mu;l nir with 1 &mu;l B0030 (rbs)
 * 3 &mu;l I14032 with 1 &mu;l B0030 (rbs)


 * Used 5 &mu;l ligation reaction to transform 50 &mu;l 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


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

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!