Brandon:Protocols/TBU gel: Difference between revisions

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*4. Denature at 75C for 8 minutes
*4. Denature at 75C for 8 minutes
*5. Transfer to ice quickly
*5. Transfer to ice quickly
*6. pre-run gel 2D well denaturing gel with Urea, in 0.5X TBE buffer. run for 10 minutes.
*6. pre-run gel in denaturing gel with Urea, in 0.5X TBE buffer. run for 10 minutes.
*7. Flush well to remove all urea. (make sure no liquid strands when done pipeting)
*7. Flush well to remove all urea. (make sure no liquid strands when done pipeting)
*8. load samples quickly, run at 200 V for 30 minutes or 250 V for 20 minutes
*8. load samples quickly, run at 200 V for 30 minutes or 250 V for 20 minutes

Revision as of 18:14, 13 March 2012

TBU gel for assaying single stranded DNA

  • 1. get gel from "probe prep only" gel box in 4 degree fridge, 2x Urea sample buffer, 10 bp ladder.
  • 2. remove plastic bottom cover (otherwise won't run)
  • 3. need for each sample:
 1X TBE urea sample buffer (2X)
 1X uL sample, (more the better)
 ladder:
 .5  uL  Invitrogen 10 bp ladder
 4.5 uL  dH20
 5.0 uL TBE urea sample buffer (2X)
  • 4. Denature at 75C for 8 minutes
  • 5. Transfer to ice quickly
  • 6. pre-run gel in denaturing gel with Urea, in 0.5X TBE buffer. run for 10 minutes.
  • 7. Flush well to remove all urea. (make sure no liquid strands when done pipeting)
  • 8. load samples quickly, run at 200 V for 30 minutes or 250 V for 20 minutes
  • 9. remove gel from its container, put into pipet box sized box in ~10-20 mL of .5x TBE
    • add ~3 uL of SYBR gold, and put on orbital shaker for 5 minutes. (or can do 100 ml buffer, 5 uL SYBR gold to stain)
    • do in a clean tray! for a clear image..
  • 10. put into gel bio-red gel dock (imaging station)
 a. hit "EPI white" so you can see gel and position into the middle
 b. hit "TRANS UV", and then manual acquire to get pix
 c. adjust "exposure time (sec)" until you can see it gel clearly
  • 11. hit "annotate" button
 to quantitate:
 a. volume tab -> volume rect tool
 b. hold control to copy
 c. do the lower sizes?
 d. reports tab, volume analysis report
 e. export as jpeg file
 otherwise just save
  • 12. dump gel in blue "gel disposal" container, and TBE w/ SYBR gold in blue capped container.