for
Alu Polymorphism
Please
note: The
checklist assumes that you are familiar
with
basic techniques and just need to know what the steps are!Ý
Refer to the complete protocols if you need more information.
Revised May
2002
Part I:Ý Cheek
Cell DNA Isolation
___ 1.Ý Swab cheeks (both sides) with sterile cotton swab.
___ 2.Ý Break or cut off the end of the swab and place into a microfuge tube.
___ 3.Ý Add 400 μL phosphate buffered saline (PBS).
___ 4.Ý Add 400 μL buffer AL.
___ 5.Ý Add 20 μL proteinase K, then vortex immediately for 15 seconds.
___ 6.Ý Incubate at 56οC for 10 minutes.
___ 7.Ý Add 400 μL pure ethanol (ETOH; 190-200 proof), then vortex immediately for 15 seconds.
___ 8.Ý Transfer 700 μL into a QIAmp spin column seated in a 2 mL collection tube.
___ 9.Ý Centrifuge at 8,000 RPM for 1 minute, then discard the flow-through (in the collection tube).
___ 10.Ý Add 500 μL buffer AW1 to the spin column.
___ 11.Ý Centrifuge at 8,000 RPM for 1 minute, then discard the flow-through.
___ 12.Ý Add 500 μL buffer AW2 to the spin column.
___ 13.Ý Centrifuge at 14,000 RPM for 3 minutes.
___ 14.Ý Carefully remove the spin column from the collection tube and discard the collection tube.
___ 15.Ý Place the spin column in a new microfuge tube.
___ 16.Ý Add 150 μL buffer AE to the spin column.
___ 17.Ý Incubate at room temperature for 1 minute.
___ 18.Ý Centrifuge at 8,000 RPM for 1 minute.Ý The solubilized DNA is now at the bottom of the microfuge tube.
___ 19.Ý Remove and discard the spin column.
___ 20.Ý Label the microfuge tube and proceed to Part II (or store samples in freezer)
Part II:Ý
Polymerase Chain Reaction (PCR)
Prepare reaction using a PCR bead, 10 μL DNA extract, and 15 μL primer mix (total reaction volume is 25 μL.).Ý Start the reaction using the 'cheek' program in the thermocycler.Ý
Part III:Ý Agarose
Gel Electrophoresis
Electrophorease PCR products on 2% w/v agarose gel using TAE buffer (75V for ~35 minutes).Ý Don't forget to use a 100 bp ladder.Ý Stain with ethidium briomide and visualize.