User:Jeffrey Kim/Notebook/Halogenated natural products from uncultured bacteria/2009/01/07

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454 Prep

  • 454 Prep yielded interesting results that did not deviate too much from our previous run. Summary: All titration points (1, 1/2, 1/4, 1/8 template per bead concentration) yielded essentially the same bead recovery rates (3.2%, 3.2%, 5.2%, 5.2%) with no linearity. This speaks toward the prep being f-ed up by something that is in the sample. Potentially a contaminant but nothing showed up in the DNA chip analysis of the products (no small products, no primer dimer, no consistent peak in each sample apart from what we were looking for). We QC'ed MID 1-3 but ran with 2, 3 due to low coverage in the last round.
  • We are planning on doing a blank control to make sure it's not in our amplification mix and it is not in the emulsion oil. We will also take a look at the previous run to see if the data yields any consistent band.

TAR cloning progress

  • TAR cloning is proceeding as planned. 50 ml ON cultures (pLLx8 pLLx13 amp, tet res) were spun down at 4000xg 20' in conicals and miniprepped in 4 separated aliquots (qiagen). DNA yield is ~250ng/ul for a total of 62.5ug DNA of each plasmid. Plenty for templated PCR prep for the TAR cloning project. The yeast strains are still growing at 30C...giving it one more day because colonies are quite small.

Glycopeptide Project

  • Q87 M13 end primer set 1 yielded a clean PCR product at 63, 65C. Ran a UTAH library screen using 65C and standard thermopol conditions for 35 cycles. Control looked like $(#) but Pools J, M, N, and Q yielded nice clean PCR product of the right size. Pools M and N have contaminant clones so pool J was setup for row and column screens in order to gel purify and chase down the clone for isolation. This is being pursued because we ran into unexpected tailoring modules on the end of Q87 where the oxidative coupling enzymes should have resided. A more detailed analysis of the sequencing data will be performed in order to parse out any reasons why this could have appeared.
  • ZB118 T7 end primer sets will be re-assessed to see if we need to go after that end as well. It truly looks like we hit the end of the gene cluster on that side due to the high degree of homology with known g-peptide producing biosynthetic pathways...but...because things are getting interesting on the other end, we may go after that as well.



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