Melbourne/Lab Notebook gv 1


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June 2007: Create Glycerol stocks of gas vesicle plasmids and cells supplied by Maura Cannon

  • Make LB
  • Make TE
DNA concentrations in ng/uL
Colony Sample no: 1 2 3
pNL26P (from Agar) 20 31 33
pNL26T (from plasmid) 29 29 31
pNL29P (from Agar) 28 27 28
pNL29T (from plasmid) 36 35 31
  • Digest 26uL of each in Buffer2 with 1uL HindIII (20U) at 37degC for 1h35min. (30uL reaction)
  • Prepared 20 lane 100mL agarose gel
  • Loaded 20uL of digest samples in the following lane order
    • All pNL29 (8983bp) minipreps were cut into 3 fragments as expected (4852bp,3479bp,652bp)
    • All pNL26 (10318bp) minipreps were cut into 4 fragments as expected (4852bp,3479bp,1335bp,652bp)
    • See Gell
  • Performed serial trippling dilution of 1Kb+ marker to determine sensitivity of Ethidium bromide visualisation on Gel. Based on 1650bp band (76ng in 20uL of marker) we can see about 10ng of Dna. Hence to see 652bp band in pNL26(10318bp) we needed to digest at leat 10ng x 10318/652 /20ng/ul=8uL of minipreped pasmid , so we used 26uL above.
  • Due to low yeild Craig performed a second overnight culteure and used Alkaline Lysis miniprep protocol to obtain 10.9mg/ml pNL29 and 9.9mg/ml pNL26, however these were not used.