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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 Buffer2.5uL
0.1M MgCl20.375ul
Primer1ul
Taq1ul
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
# 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
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