Primary Amplification of Genomic DNA using DOP - PCR

互联网2013-12-26

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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