SNuPE Protocol

Make sure to wear gloves, eye goggles and a lab coat during the procedure!

Instructions

  1. Make sure that there is enough dCTP and TTP (or dGTP and dATP) in the hot room
  2. Set up your PCR master mix.  Remember that you need to do a reaction for dCTP and TTP for each sample
    1. 10xPCR Buffer  2.5uL
    2. 0.1M MgCl2  0.375ul
    3. Primer  1ul
    4. Taq  1ul
    5. dH20  - enough to bring final volume to 25 ul
  3. Aliquot mix into each tube and place rack on ice
  4. Add your DNA to tubes (DNA + dH20 needs to be 19.625 ul)
  5. Take PCR into hot room and add 0.5 ul of appropriate nucleotide into tube
  6. Cap and mix by short vortex
  7. Run SNuPE program on PCR machine Ð make sure lids are on TIGHT
    1. 95º for 1 min
    2. 50º for 2 min
    3. 72º for 1 min
    4. 4º for 5 min
  8. While this is on going, prepare spin columns.  Cut off tops of eppies for collection
  9. Briefly vortex column and then snap off bottom and turn top a bit.  Place in collection eppies
  10.  Spin for 1 min at 700g in centrifuge
  11. Prepare screw top tubes for columns ­ label tops and add 5ul of loading dye to bottom of tubes
  12. Place columns in screw top tubes ­ will use these for collection of SNuPE product
  13. When PCR is done, put on ice and bring back to hot room
  14. Take off tops and discard.  Add 1ul 0.5M EDTA to stop the reaction. Place new tops on PCR tubes
  15. Bring back to PCR machine and run the M program
    1. 95º for 10 min
    2. 4º for 5 min
  16. Chill on ice
  17.  Remove tops and transfer 25 ul to the spin column.  Spin for 2 min at 2.3 RPM in hot room centrifuge
  18. Discard column and place new top on tube.  Vortex to mix up loading dye.
  19. SNuPEs can be placed at 4º until ready to run
  20. For the gel, use this recipe
# of Gels 1 2
40% Acrylamide 3.75 mL 5.625 mL
5x TBE Buffer 1 mL 1.5 mL
dH20 5.25 mL 7.875 mL
Urea 4.2 g 6.3 g
10% APS 100 uL 200 uL
TEMED 10ul 20ul
  1. Mass Urea and dissolve in H2O first.  Make sure the Urea is all dissolved before moving on.
  2. Set up the gel apparatus ­ make sure that you place some parafilm underneath to prevent the gel from leaking
  3. After loading, place in the chamber and pour in 0.5X TBE as the running buffer
  4. Clean out the wells with a P1000
  5. Load your samples with the gel loading tips ­ 10-15uL is enough for one gel.  This leaves you some leftover in case you need to run another gel.
  6. Run for about 40-45 minutes at 150volts.  About 2/3rds of the way down
  7. Take apart gel apparatus and place gel on 3M Whatman paper. Cover with saranwrap
  8. Place in the gel drier ­ dry at 80º for at least 1.5 hours
  9. Place in autorad or phosphorimager cassette