Brandon:Protocols/TBU gel: Difference between revisions
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==TBU gel for assaying single stranded DNA== | ==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 | *1. get gel from "probe prep only" gel box in 4 degree fridge, 2x Urea sample buffer, and low mass ladder if normal. (10 bp ladder if probes) | ||
*2. remove plastic bottom cover (otherwise won't run) | *2. remove plastic bottom cover (otherwise won't run) | ||
*3. | *3. for each sample: | ||
1.0 uL sample or Invitrogen low mass ladder | |||
4.0 uL dH20 | |||
4. | |||
5.0 uL TBE urea sample buffer (2X) | 5.0 uL TBE urea sample buffer (2X) | ||
*4. Denature at 75C for 8 minutes | *4. Denature above sample solutions and ladder at 75C for 8 minutes | ||
*5. Transfer to ice quickly | *5. Transfer to ice quickly | ||
*6. pre-run gel | *6. pre-run gel in 0.5X TBE buffer. run for at least 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 180V for 45 minutes, or 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 | *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) | **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) |
Latest revision as of 22:03, 17 April 2012
TBU gel for assaying single stranded DNA[edit]
- 1. get gel from "probe prep only" gel box in 4 degree fridge, 2x Urea sample buffer, and low mass ladder if normal. (10 bp ladder if probes)
- 2. remove plastic bottom cover (otherwise won't run)
- 3. for each sample:
1.0 uL sample or Invitrogen low mass ladder 4.0 uL dH20 5.0 uL TBE urea sample buffer (2X)
- 4. Denature above sample solutions and ladder at 75C for 8 minutes
- 5. Transfer to ice quickly
- 6. pre-run gel in 0.5X TBE buffer. run for at least 10 minutes.
- 7. Flush well to remove all urea. (make sure no liquid strands when done pipeting)
- 8. load samples quickly, run at 180V for 45 minutes, or 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.