User:Jeffrey Kim/Notebook/Halogenated natural products from uncultured bacteria/2009/01/06

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TAR cloning project

  • YPD plates and agar (20g/l) for Yeast maintenance.
  1. Dissolve 10g of BactoYeast extract in 500ml water
  2. Dissolve 20g of BactoPeptone in the above solution
  3. Dissolve 20g Dextrose in the above solution
  4. q.s. to 1000ml with water
  • Yeast strain (S.Lory - Uracil deficient) struck out on YPD plates and placed in 30C incubator for colony selection.
  • pLLX8 and pLLX13 both grown in (amp tet respectively) LB 10ml cultures. 50ul used to innoculate a 50ml culture for ON growth at 37C

454 Training

  • 454 training initiated with mRNA pico chip analysis. We will proceed with a ribogreen quantitation and then a dilution series into emulsions for emPCR setup overnight. The thermal cycler may be a problem in this setup so we may test a different thermal cycler.
  • Ribogreen QC did not go well (titration curve gave ambiguous results) so we went with the previous values from the same prep. MID's 1-3 quantitated and MID's 2, 3 used for the dilution series at (1, 0.5, 0.25, (1/8)) copies of DNA template per bead for emPCR.
  • Thermal cycler conditions looked fine and were setup for an overnight run

glycopeptide project

  • Q87 M13 primer sets 1 and 2 were tested in a gradient against the Q master pool and Q87 single isolate as a control. 35 cycles using standard Taq buffer and conditions looked good for primer set 1. Will screen the UTAH library using optimized conditions to go after the M13 end. Will also analyze and design primers against the T7 end of ZB118 in order to recover additional ends of the pathway.


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