Brandon:Protocols/DNA gel: Difference between revisions

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>Bsos
(Created page with '==TBE gel for validation that primers amplified the target== *1. get gel from 4 degree fridge, NOT 1.5 sized gel, want normal sized ones *2. remove plastic bottm cover (otherwise…')
 
>Bsos
 
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==TBE gel for validation that primers amplified the target==
==TBE gel for validation that primers amplified the target==
*1. get gel from 4 degree fridge, NOT 1.5 sized gel, want normal sized ones
*1. get gel from 4 degree fridge, NOT 1.5 sized gel, want normal sized ones and LM ladder, and 6X loading dye.
*2. remove plastic bottm cover (otherwise won't run)
*2. remove plastic bottom cover (otherwise won't run)
*3. need for each sample:
*3. need for each sample:
   3 uL blue gel loading dye (6x)
   3 uL blue gel loading dye (6x)
   2 uL dIH2O (or .5x TBE buffer)
   2 uL dIH2O (or .5x TBE buffer)
   1 uL sample, its lots as determined by nano, or 3-5 uL if very little DNA
   1 uL sample, its lots as determined by nano, 50 ul reactions use 2-3 uL, 100 uL reactions use 3-4 uL
 
*4. mix ladder, and samples in their own mixtures on parafilm, then load into gel.
*4. mix on together on parafilm, then load into gel.
*5. run for 20 minutes at 250 volts or 30 minutes at 200 volts.
*5. run for 20 minutes at 250 volts or 30 minutes at 200 volts.
*6. remove gel from its container, put into pipet box sized box in ~10-20 mL of .5x TBE
*6. remove gel from its container, put into pipet box sized box in ~10-20 mL of .5x TBE

Latest revision as of 23:22, 5 August 2011

TBE gel for validation that primers amplified the target[edit]

  • 1. get gel from 4 degree fridge, NOT 1.5 sized gel, want normal sized ones and LM ladder, and 6X loading dye.
  • 2. remove plastic bottom cover (otherwise won't run)
  • 3. need for each sample:
 3 uL blue gel loading dye (6x)
 2 uL dIH2O (or .5x TBE buffer)
 1 uL sample, its lots as determined by nano, 50 ul reactions use 2-3 uL, 100 uL reactions use 3-4 uL
  • 4. mix ladder, and samples in their own mixtures on parafilm, then load into gel.
  • 5. run for 20 minutes at 250 volts or 30 minutes at 200 volts.
  • 6. 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 rotator for 5 minutes
  • 7.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
  • 8. 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
  • 9. dump gel in blue "gel disposal" container, and TBE w/ SYBR gold in blue capped container.