Primary Amplification of Genomic DNA using DOP - PCR
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	Reagents
	Agarose, Ultrapure
	Gibco, BRL, Cat. no. 15510-027
	10X Buffer, and
	Perkin Elmer (Part no. N808-0010)
	Template DNA
	Use 50-100 ng for each reaction
	Ethidium Bromide
	Research Genetics, Cat. no. 750007
	Loading buffer, 5X
	Quality Biological, Cat. no. 51-026-030
	MgCl2, 25 mM
	Perkin Elmer (Part no. N808-0010)
	100 mM dNTP nucleotides
	dGTP
	Boehringer Mannheim, 1051466
	dCTP
	Boehringer Mannheim, 1051458
	dATP
	Boehringer Mannheim, 1051440
	dTTP
	Boehringer Mannheim, 1051482
	Primer “UN1”
	Midland Certified Reagent Co. Telenius 6MW
	[5’-CCGACTCGAGNNNNNNATGTGG-3’]
	Taq DNA polymerase, 5 U/μl
	Fermentas Life Sciences, Cat. Epo282 500U
	TAE buffer, 10X
	Advanced Biotechnologies, Cat. no.08-514-001
	Sterile water (H20)
	Molecular grade sterile distilled water 
	Materials and Equipment
	PCR Thermocycler
	Gel system and power source
	PCR tubes
	Preparation
	1X TAE buffer
	Dilute the 10X TAE with dH20, 1:10 to make 1X TAE
	Dissolve 1 g of agarose in100 ml 1X TAE buffer by warming the solution
	Stock dNTP 2mM
	μl mM final
	dGTP 10 0.2
	dCTP 10 0.2
	dATP 10 0.2
	dTTP 10 0.2
	dH20 460 -
	total 500
	• Autoclave PCR microcentrifuge tubes (0.5 ml size), 2 ml
	microcentrifuge tubes, and pipet tips (10, 200, and 1000 μl).
	• Sterilize pipettes (using UV) to be used for PCR and only use that set
	for PCR (can use Stratalinker or UV light source from tissue culture
	hood).
	• Before starting, make sure that the work space is cleaned with ethanol
	and that it is located in a low traffic area. Use the tissue culture hood
	to maintain sterility if you cannot find a corner to work in.
	Procedure
	1. Label each 0.5 ml tube, being careful to only handle the outside of the tube.
	2. Add appropriate volume of DNA to each PCR tube, close, and set aside into
	ice or 4°C until the reaction mix is ready to aliquot
	3. The order for pipetting the reaction mix is as follows: dH20, buffer, MgCl2,
	dNTP, and primer. The reaction mix for one reaction is as follows (multiply
	each by the number of reactions):
	Primer 4 μl
	Genomic DNA Y μl (Note 1)
	10X buffer 10 μl
	MgCl2 25mM 8 μl
	dNTP 10 μl
	Sterile dH20 X μl (Note 2)
	4. Pipette 96 μl of the reaction mix into each PCR tube (change tips between
	each tube) and put on ice.
	5. Vortex the tubes, spin quickly (few seconds, not higher than 5000 rpm), and
	place on ice. At this point take out the Taq enzyme, mix carefully (tap with
	finger), spin quickly, then add 1 μl (for each 100 μl) (Note 3) to each reaction.
	Vortex the tubes, spin quickly, and put back on ice.
	6. Vortex each tube, spin quickly, and put them all into the PCR machine and
	run using the appropriate PCR program (Note 4).
	7. After completion, remove the tubes from the PCR machine, vortex, spin
	quickly, and place on ice. Mix 0.8 μl of 5X DNA loading buffer with a 2 μl
	aliquot from each reaction to run on a 1% agarose gel. The resulting smear
	migrates around 500 bps (Note 5).
	Notes
	1. The volume of genomic DNA depends on the concentration that you have
	available.
	2. The volume of water to add depends on the volume of DNA that is added, and
	the total reaction volume should be equal to 100 μl.
	3. The Taq polymerase should always be added last, and since it is more viscous
	and sticky, it needs to be mixed well before each use. Use the pipet tip you
	are about to draw with to gently stir the contents as you draw up the enzyme.
	4. PCR program (runs approximately 7 hours):
	Step Temperature (°C) Minutes
	1 (initial denaturation). 93 10
	2 94 1
	3 30 1.5
	4 ramp 30-70 3
	5 72 3
	6 repeat steps 2-5, 4 times
	7 94 1
	8 62 1
	9 72 3 + 1 second/cycle
	10 repeat steps 7-9, 34 times
	11 72 10
	12 4 ∞
	5. 1% agarose gel
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