User:DavidRamos/Lab/2006-6-14

From OpenWetWare

Jump to: navigation, search

June 14th

Morning

Plasmid Miniprep

  • lac operon promoter
    • R0010
  • promoter and GFP
    • E0241
  • GFP
    • E7104
  • DNA Miniprep of transformant colonies
    • Out of 5mL of liquid culture, reserved 1mL for gylcerol+freeze and 4mL other
    • followed QIAprep Miniprep Kit for Microcentrifuge directions
      • Eluted with warm dH20
      • Put at 40C for ~2 min to evaporate ethanol before elution
      • Forgot to label after elution --> don't know what is what
        • Sol'n: During digest will have to run PCR, can tell R0010 from rest, but E7104/E0241 is only different by 30bp; if doesn't work can flip and try two experiments.
      • Nanodrop demonstration
Nanodrop results
  -1: 31.2ng/uL 260/280:1.75 260/230:1.92
  -2: 38.5ng/uL 260/280:1.83 260/230:1.87
  -3: 33.8ng/uL 260/280:1.70 260/230:1.44

PROBLEM: Messed up labeling of the plasmids! To diagnose, ran a 15min e-Gel to find out which is R0010.

  • Digestion of vector/insert
    • Digested R0010 (200bp cutout) as vector at S and P site.
      • .5uL Spe1, .5uL Pst1
      • 11uL h20
      • 2.5uL 10X BSA
      • 2.5uL #2 NebBuffer
      • 8uL DNA
    • Digested other 2 (~900bp cutout) as insert at X and P site.
      • .5uL Xba1, .5uL Pst1
      • 11uL h20
      • 2.5uL 10X BSA
      • 2.5uL #3 NebBuffer
      • 8uL DNA
    • Incubate @ 37C for 1h
  • Phosphatase
    • 80C@15min to kill enzyme activity
    • Used CIP (1 unit) into the R0010, 1h@37C
  • Run on 1% agarose gel
  • Image, Cutout, and Purify
    • Can isolate the three from the gel

Result

"results of PCR"

 Ladder=1kb+
 Lane 1=R0010 (#1)
 Lane 2=E0241 (#2)
 Lane 3=E7104 (#3)
Personal tools