Quick Guide Lesson 1: Setting up the PCR Reactions

Quick Guide Lesson 1: Setting up the PCR Reactions
Quick Guide
Lesson 1: Setting up the PCR Reactions
1.
Label 5 PCR tubes CS, A, B, C, or D, and
include your group name or initials as well.
Place each PCR tube into a capless micro
centrifuge tube in the foam float on ice.
PCR tube
2.
Capless
tube
Using the chart below as a guide, transfer
20 µl of the appropriate template DNA into
the correctly labeled tube. Important: use a
fresh aerosol barrier pipet tip for each
DNA sample.
Label PCR tubes
Add DNA template
Add Master mix + primers
CS + your initials
20 µl Crime Scene DNA
20 µl MMP (blue)
A + your initials
20 µl Suspect A DNA
20 µl MMP (blue)
B + your initials
20 µl Suspect B DNA
20 µl MMP (blue)
C + your initials
20 µl Suspect C DNA
20 µl MMP (blue)
D + your initials
20 µl Suspect D DNA
20 µl MMP (blue)
3.
Transfer 20 µl of the blue MMP (master mix +
primers) into each of the 5 PCR tubes
containing template DNA. Pipet up and down
to mix. Cap each tube after adding blue
MMP. Important: use a fresh aerosol
barrier pipet tip each time. Immediately
cap each tube after adding MMP.
4.
Place your capped PCR tubes in their adaptors
on ice.
5.
When instructed to do so, place your tubes in
the thermal cycler. Your instructor will program
the thermal cycler for PCR.
27
Master mix +
primers
Lesson 2: Electrophoresis of PCR
Products
1.
Set up your gel electrophoresis equipment as
instructed.
2.
Obtain your 5 PCR tubes from the previous
lesson. Place your PCR tubes in capless
tubes and pulse-spin in a balanced
microcentrifuge for a few seconds to collect
all liquid to the bottom of the tube.
3.
Transfer 10 µl of Orange G loading dye (from
the tube labeled 'LD') into each of your PCR
tubes. Pipet up and down to mix, and
pulse-spin to collect liquid in the bottom of the
tube.
4.
Place an agarose gel in the electrophoresis
apparatus. Check that the wells of the
agarose gel are near the black (–) electrode
and the base of the gel is near the red (+)
electrode.
5.
Fill the electrophoresis chamber with enough
1x TAE buffer to cover the gel. This will
require ~275 ml of 1x TAE buffer.
Centrifuge
–
+
6.
Using a clean tip for each sample, load 20 µl
of the samples into 6 wells of the gel in the
following order:
Lane
1
2
3
4
5
6
Sample
Allele Ladder
Crime Scene
Suspect A
Suspect B
Suspect C
Suspect D
Load volume
20 µl
20 µl
20 µl
20 µl
20 µl
20 µl
28
7.
Secure the lid on the gel box. The lid will
attach to the base in only one orientation: red
to red and black to black. Connect the
electrical leads to the power supply.
–
+
8.
Turn on the power supply and electrophorese
your samples at 100 V for 30 minutes.
9.
Stain in Fast Blast DNA stain. Refer to the
Student Manual for specific instructions.
29
Bio-Rad
Laboratories, Inc.
Web site www.bio-rad.com USA (800) 4BIORAD Australia 02 9914 2800 Austria (01)-877 89 01 Belgium 09-385 55 11 Brazil 55 21 2527 3454
Canada (905) 712-2771 China (86 21) 6426 0808 Czech Republic + 420 2 41 43 05 32 Denmark 44 52 10 00 Finland 09 804 22 00
France 01 47 95 69 65 Germany 089 318 84-0 Greece 30 210 777 4396 Hong Kong (852) 2789 3300 Hungary 36 1 455 8800
India (91-124)-2398112/3/4, 5018111, 6450092/93 Israel 03 951 4127 Italy 39 02 216091 Japan 03-5811-6270 Korea 82-2-3473-4460
Latin America 305-894-5950 Mexico 55-52-00-05-20 The Netherlands 0318-540666 New Zealand 64 9 415 2280 Norway 23 38 41 30
Poland + 48 22 331 99 99 Portugal 351-21-472-7700 Russia 7 095 721 1404 Singapore 65-64153188 South Africa 00 27 11 4428508
Spain 34 91 590 52 00 Sweden 08 555 12700 Switzerland 061 717 95 55 Taiwan (886 2) 2578 7189/2578 7241 United Kingdom 020 8328 2000
Life Science
Group
Bulletin 5292 US/EG
Rev A
05-0416
0405
Sig 1204
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