Brandon:Protocols/gel size selection: Difference between revisions

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>Bsos
(Created page with '==gel size selection protocol== *cut band from gel, extract DNA for downstream analyses/applications *1. run gel as normal [http://genome-tech.ucsd.edu/LabNotes/index.php/Brand…')
 
>Bsos
 
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*cut band from gel, extract DNA for downstream analyses/applications
*cut band from gel, extract DNA for downstream analyses/applications


*1. run gel as normal [http://genome-tech.ucsd.edu/LabNotes/index.php/Brandon:Protocols/DNA_gel PAGE gel protocol]. 2-3 ug DNA per well if using the big wells (4-wells per plate). usually 2 wells per 25 uL PCR reaction when using the 5 well gel. <1 ug per well when using the smaller wells. with 1 mm gel
*1. run gel as normal [http://genome-tech.ucsd.edu/LabNotes/index.php/Brandon:Protocols/DNA_gel PAGE gel protocol].
** in 12 well gel, 1.0 mm: <1 ug max per gel, or 80 ng/well
** in 5 well gel, 1.0 mm: 1.0-1.3 ug total DNA max per gel , 250-300 ng/well (2 wells per 25 uL PCR reaction)
** in 5 well gel, 1.5 mm: 1.5 -2.0 ug total DNA max
** in 2 well gel, 1.5 mm: 2 ug total DNA max
 
  Notes:
  Notes:
rinse gel holders with clean TE before use
  6X dye (5 uL for 25 uL sample)
  6X dye (5 uL for 25 uL sample)
  dilute with TE if needed
  dilute with TE if needed
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  put on shaker for 3-4 minutes
  put on shaker for 3-4 minutes


*2. poke hole into .5 mL tube, put into 1.5 mL tube. (cut band goes into .5 mL tube)
*2. poke hole into .5 mL tube with 22 gauge needle, put into 2.0 mL tube. (cut band goes into .5 mL tube)


*3. cut out band in dark room, use new scalpel for each sample. don't expose to UV for to long. wear glasses. use shield. place gel into .5 mL tube prepared above.
*3. cut out band in dark room, use new scalpel for each sample. put syran wrap over Blue light illuminator box, then put gel on top. wear glasses. use shield. place gel into .5 mL tube prepared above.


*4. centrifuge (.5 mL tube in 1.5 mL tube) for 3 minutes at 15k RPM. (gel sheared)
*4. centrifuge (.5 mL tube in 2.0 mL tube) for 3 minutes at 15k RPM. (gel sheared). image cut gel.


*5. discard .5 mL tube. add 450 uL 1X TE to 1.5 mL tube.
*5. discard .5 mL tube. add 450 uL 1X TE to 1.5 mL tube.
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*6. Shake at 37C for 45 minutes.
*6. Shake at 37C for 45 minutes.


*7. centrifuge at 13k RPM for 1 minute. pipet supernatent into blue filter column (nanosep MF .2 uM filter tubes).
*7. pipet supernatent into blue filter column (nanosep MF .2 uM filter tubes).


*8. centrifuge blue filter tube at 13K RPM for 1 minute. pipet rest of gel pieces into blue filter tube.
*8. centrifuge blue filter tube at 13K RPM for 1 minute. pipet rest of liquid in gel pieces into blue filter tube.


*9. centrifuge at 13K RPM for 1 minute.
*9. centrifuge at 13K RPM for 1 minute. repeat so no liquid above filter


*10. add 2.5 volumes 100% EtOH, .1 volumes 3M NaoAc, and 1/300 volumnes glycol-blue to solution obtained.
*10. add 2.5 volumes 100% EtOH, .1 volumes 3M NaoAc, and 1/300 volumnes glycol-blue to solution obtained.
  In this case:
  In this case: (ADD IN THIS ORDER)
1  ml 100% EtOH
  40  uL NaOAc
  40  uL NaOAc
  1.3 uL glycolblue
  1.3 uL glycolblue
1  ml 100% EtOH


*11. Switch solution to round bottom 1.5 mL tube.
*11. Switch solution to round bottom 1.5 mL tube. invert tube to mix.


*12. put into -80C for 15 minutes.
*12. put into -80C for 15 minutes. cool centrifuge to 4C.


*13. centrifuge at 4C for 20 minutes. chill 75% EtOH.
*13. centrifuge at 4C for 20 minutes at 10K RPM (10K RCF, max speed). chill 75% EtOH.


*14. blue pellet should be visible. discard supernatant.
*14. blue pellet should be visible. discard supernatant.
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*16. Centrifuge at 14K RPM for 5 minutes at RT.
*16. Centrifuge at 14K RPM for 5 minutes at RT.


*17. discard supernatent, dry pellet for 5 minutes.
*17. discard supernatent, pipet out EtOH with gel tips, invert tube on kimwipes, dry pellet for 5 minutes.


*18. add 15 uL nuclease free H2O to pellet, pipet up and down to resuspend.
*18. add 5 to 20 uL nuclease free H2O to pellet, pipet up and down to resuspend.
  store in 4C
  store in 4C
  can quantitate in PAGE gel.
  can quantitate in PAGE gel.

Latest revision as of 19:47, 21 December 2015

gel size selection protocol[edit]

  • cut band from gel, extract DNA for downstream analyses/applications
  • 1. run gel as normal PAGE gel protocol.
    • in 12 well gel, 1.0 mm: <1 ug max per gel, or 80 ng/well
    • in 5 well gel, 1.0 mm: 1.0-1.3 ug total DNA max per gel , 250-300 ng/well (2 wells per 25 uL PCR reaction)
    • in 5 well gel, 1.5 mm: 1.5 -2.0 ug total DNA max
    • in 2 well gel, 1.5 mm: 2 ug total DNA max
Notes:
rinse gel holders with clean TE before use
6X dye (5 uL for 25 uL sample)
dilute with TE if needed
normal for ladder
use clean pipet box top to hold gel/solution for SYBR gold (2 uL) staining, and clean TE buffer
put on shaker for 3-4 minutes
  • 2. poke hole into .5 mL tube with 22 gauge needle, put into 2.0 mL tube. (cut band goes into .5 mL tube)
  • 3. cut out band in dark room, use new scalpel for each sample. put syran wrap over Blue light illuminator box, then put gel on top. wear glasses. use shield. place gel into .5 mL tube prepared above.
  • 4. centrifuge (.5 mL tube in 2.0 mL tube) for 3 minutes at 15k RPM. (gel sheared). image cut gel.
  • 5. discard .5 mL tube. add 450 uL 1X TE to 1.5 mL tube.
  • 6. Shake at 37C for 45 minutes.
  • 7. pipet supernatent into blue filter column (nanosep MF .2 uM filter tubes).
  • 8. centrifuge blue filter tube at 13K RPM for 1 minute. pipet rest of liquid in gel pieces into blue filter tube.
  • 9. centrifuge at 13K RPM for 1 minute. repeat so no liquid above filter
  • 10. add 2.5 volumes 100% EtOH, .1 volumes 3M NaoAc, and 1/300 volumnes glycol-blue to solution obtained.
In this case: (ADD IN THIS ORDER)
40  uL NaOAc
1.3 uL glycolblue
1   ml 100% EtOH
  • 11. Switch solution to round bottom 1.5 mL tube. invert tube to mix.
  • 12. put into -80C for 15 minutes. cool centrifuge to 4C.
  • 13. centrifuge at 4C for 20 minutes at 10K RPM (10K RCF, max speed). chill 75% EtOH.
  • 14. blue pellet should be visible. discard supernatant.
  • 15. add 700 uL of ice-cold 75% EtOH. Invert tube 4-5 times.
  • 16. Centrifuge at 14K RPM for 5 minutes at RT.
  • 17. discard supernatent, pipet out EtOH with gel tips, invert tube on kimwipes, dry pellet for 5 minutes.
  • 18. add 5 to 20 uL nuclease free H2O to pellet, pipet up and down to resuspend.
store in 4C
can quantitate in PAGE gel.