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 Crandall Lab Protocols

The Crandall Lab Crandall Lab Protocols

Tissue Digestion / Extraction

DNA Extraction Check / Dilution

PCR Reactions

PCR Cleanup

Sequencing Reactions

Sephadex Cleanup

Tissue Digestion / Extraction

 

Tissue Digestion / Extraction: Cell lysis protocol
  1. Cut up tissue (~25mg) into small pieces
  2. Blot tissue to remove excess ethanol. Place in labeled 2.0mL tubes
  3. Add 800ul Cell Lysis buffer
  4. Add 9uL Proteinase K (20mg/ml)
  5. Vortex samples 15 seconds to mix
  6. Incubate @ 55º until tissue is completely lysed (over night if needed) vortexing samples occasionally
  7. Vortex samples then spin tubes at 13000rpm for 1 minute.
    1. Undigested debris will be pelleted
  8. Transfer supernatant to new 2.0mL tube without disturbing pellet.
  9. Add 180 uL of 5M NaCl and vortex well.
    1. Solution will become frothy.
  10. Spin tubes at 13000rpm for 5 minutes.
    1. Salted out debris will pellet
  11. Transfer supernatant to cryotubes (screw-cap)
  12. Add 420uL ice-cold isopropanol (2-propanol) to supernatant
    1. Mix slowly by inversion 5-10 times DO NOT VORTEX
    2. DNA fibers may be seen at this time
  13. Spin tube at 13000 rpm for 10 minutes.
    1. DNA pellet should be visible
  14. Pour out supernatant
  15. Add 400uL 70% ethanol to wash DNA pellet.
    1. Wash 20 minutes on cell rotator at room temp
  16. Spin tubes at 13000 rpm for 5 minutes and pour out ethanol carefully! Pellet may be loose. If pellet is loose pipette ethanol out being careful to not disturb the pellet.
  17. Dry DNA pellet in speed vac on High for 10 minutes.
  18. Resuspend pellet in TLE, TE or purified H2O
    1. If small pellet add ~ 50uL
    2. If large pellet add ~ 100uL
  19. Let tubes stand at room temp overnight

 

 DNA Digestion / Extraction: Qiagen DNeasy kit protocol

  1. Cut up tissue (~25mg) into small pieces
  2. Blot tissue to remove excess ethanol. Place in labeled 2.0mL tubes
  3. Add 180uL buffer ATL
  4. Add 20uL Proteinase K
  5. Mix by vortexing
  6. Incubate @ 55º on cell rotator until tissue is completely lysed (over night if needed) vortexing samples occasionally
  7. Vortex samples 15 seconds
  8. Add 200uL buffer AL, mixing thoroughly by vortex
  9. Incubate at 70ºC for 10 minutes
  10. Add 200uL ethanol (96-100%), mixing thoroughly by vortex
  11. Pipette the mixture into DNeasy spin columns placed in 2ml collection tube
  12. Centrifuge at 8000rpm for 1 min
  13. Discard flow-through from collection tube
  14. Place spin column in new collection tube (old collection tubes can be reused if needed)
  15. Add 500ul buffer AW1
  16. Centrifuge at 8000rpm for 1 minute
  17. Discard flow-through from collection tube
  18. Place spin column in new collection tube
  19. Add 500ul buffer AW2
  20. Centrifuge at full speed for 3 minutes
  21. Discard flow-through and collection tube (make sure not to have the spin column touch the flow-through, the membrane needs to be dry for the next step.
  22. Place spin column in clean 2.0ml tube
  23. Pipette 200ul buffer AE onto membrane
  24. Incubate at room temp for 1 minute
  25. Centrifuge at 8000rpm for 1 minute

 

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DNA Extraction Check / Dilution

  1. Make 1.5% agarose gel
  2. Pipette small amount of bromophenol blue dye onto a piece of parafilm (~ 1ul)
  3. Add 2-3ul of DNA extraction to dots of dye (2-5ul of PCR reactions)
  4. Load samples into wells
  5. Run samples at 150V until dye runs 1-2cm from wells
  6. Take tray out of rig and take picture with UV camera
  7. Dilute samples according to sample brightness

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PCR Reactions

 

Check PCR Reactions

Prepare 1.5% agarose gel

Run according to DNA Check (above)

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PCR Cleanup

 

PCR Cleanup Millipore Plates
  1. Bring total PCR volume up to 100ul
  2. Transfer all 100ul of diluted PCR reaction to a Millipore 96 well clean up plate
  3. Tape up wells not being used
  4. Put plate on vacuum manifold for 10 minutes until wells are empty, making sure pressure gauge is at -24 in Hg
    1. Wells will appear shiny, so they will look slightly wet all the time.
  5. Blot bottom of plate on paper towel to remove excess water
  6. Resuspend DNA with 30ul water
  7. Place plate on shaker / mixer for 10 minutes
  8. Pipette product out of wells and transfer to labeled tube or plate.
 Gene Clean Method for cutting bands out of gels.
  1. Run PCR product on 1.0% Agarose gel using large comb
  2. Run samples on gel at no more than 0.12Amps, until dye is half way down gel
  3. Label 2.0 ml tubes
  4. Cut out band on UV light box using razor blade (clean with water between each band) and place in labeled 2.0ml tube
  5. To each tube containing gel band add 450ml NaI
  6. Place in 55º water bath for 10minutes (to melt the gel)
  7. Add 4ul Glass Milk to each tube
  8. Vortex and let sit for 10 minutes, occasionally vortexing
    1. Glass milk will bind to DNA
  9. Spin tubes at 14000rpm for 15 seconds to pellet Glass Milk
  10. Pour out NaI
  11. Add 500ul of New Wash
  12. Vortex to resuspend pellet
  13. Centrifuge tubes at 14000rpm for 10 seconds
  14. Pour out New Wash
  15. Repeat New Wash rinse 2 more times
  16. After 3rd new Wash rinse, spin tubes again for a few seconds
  17. Remove any remaining liquid from tube
  18. Dray tubes in speed vac on High for 5 minutes
  19. Resuspend in water (15-50ul)
  20. Vortex samples carefully
  21. Place tubes in 55º water bath for 10 minutes
  22. Store in freezer

 

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Sequencing Reactions

Sequencing Reactions Big Dye v3.0

1/16th Reactions

Big Dye
0.50 uL

5x Buffer
1.75 uL

Primer (10uM)
1.00 uL

DNA (10-75ng)
2.00 uL

H2O
4.75 uL

Total Vol.
10.0 uL

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Sephadex Cleanup

of sequencing reaction

  1. Obtain Millipore multiscreen filter plates (96 well). Add a used v-bottom 96well plate to the bottom of filter plate making sure rows A-H are aligned on both plates
  2. Obtain Sephadex plate loader from DNASC
  3. Pour Sephadex on plate loader and fill all holes. Place left over sephadex back into bottle.
  4. Turn filter plate upside down and slide onto plate loader. Tap plate loader to get the sephadex to fall into the wells.
  5. Add 300ul dH2O to each well (make sure the wells are full)
  6. Let plate stand for 10-15 minutes
  7. Spin plate in centrifuge (using balance plate with water) for 2 minutes at 2500rpm. Rotate plate 180º and spin for another 4 minutes.
  8. Empty 96 well plate and place back in cupboard.
  9. Add a new (labeled) v-bottom 96 well plate to filter plate making sure rows A-H are aligned on both plates.
  10. Add 10-20ul dH2O to sequencing reaction
  11. Transfer all sequencing samples to filter plate
  12. Spin plate for 2 minutes at 2500 rpm. Rotate plate 180º and spin for another 2 minutes.
  13. Dry samples in Vacufuge at 60º for 30 minutes. Make sure to use balance plate
  14. Cover plate and mark off all unused wells.
  15. Write DNA sequence submission number on plate and cover
  16. Place plate in DNASC fridge.

 

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Last Updated: October 11, 2004