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