Allele-In-One Blood DNA
Isolation w/ Purification Kit & PCR
System, a one-step reaction using the single buffer
system plus purification for preparing DNA as PCR template.
T raditional methods for performing
genotyping using blood samples
require separation of nuclei-containing
white blood cells from red blood cells,
phenol extraction of chromosomal
DNA, and precipitation of DNA
for PCR. They are particularly not
suitable for high through-put analysis.
Allele-In-One Blood DNA Isolation &
PCR system provides a simple one-
step method for blood genomic DNA
genotyping. The Isolation Buffer
contains a combination of enzyme(s),
detergents, and other chemical
reagents that isolate enough genomic
DNA from preserved blood, clotted
blood, or isolated white blood cells
for direct use as PCR template. It is
best used in combination with Allele-
In-One PCR MasterMix, specifically
formulated to accommodate DNA
template from lysis buffer that contains
protease(s).
Allele-in-One Blood DNA Isolation
& PCR system offers a variety of
advatages including:
♦ Simple buffer system: Unique
from Allele Biotech Enzymes, this
single buffer system is the only
product that contains everything for
lysis, no need to add protease K or
any other components before use.
♦ Single step reaction: The
incubation is to be performed at a
convenient 50-55°C temperature for
0.5 hours or longer, and the lysate is
ready for PCR.
♦ Consistent: Works with extremely
high success rates and is fully
guaranteed if used in combination
with Allele-in-One PCR MasterMix
system.
♦ Lowering costs: Adds only 40
cents or less to each genotyping
sample while saving enormous
amounts of material and time from
traditional methods.
Allele Biotech-Introducing Cost Effectiveness to Research
Box 1 | Genotyping Results
Human blood samples treated with Blood DNA Isolation Buffer and 2X All-In-
One PCR MasterMix. PCR for 25 cycles and products run on a 1% agarose
gel.
Each batch of reagents is vigorously tested for consistency and stability.
Storage: -20°C and -80°C
Allele-in-One Blood DNA Isolation
buffer is suitable:
♦ Preparing genomic DNA from Protocols
human or animal blood samples for
genotyping PCR. Preserved blood samples:
1. Take 1 ml of desired number
The Blood DNA Isolation & PCR kit of blood samples, centrifuge at 3,000
includes Allele-in One PCR MasterMix, rpm in microcentrifuge tubes for
which is optimized for DNA template 5 minutes. Transfer the white-cell
prepared with the lysis buffer but can containing interface section to a new
also be used for most routine PCR set of tubes.
reactions.
2. Add 100 μl Blood DNA
Isolation Buffer to the cells, incubate
at 50-60°C, with or without agitation,
Box 2 | Product List for 15 minutes.
Blood DNA Isolation Buffer w/ 3. Purify lysate using PG
Purification Kit 100µl per reaction columns, binding buffer (AB), and
ABP-PP-BD03100 100 rxns wash buffer (AW). Elute the DNA
using the elution buffer (TE) into
ABP-PP-BD03250 250 rxns
1.5ml recovery tubes.
Blood DNA Isolation w/ Purification &
PCR Kit 10µl per reaction* 4. For each new PCR primer
ABP-PP-BD04100 100 rxns pair, use 1 μL of the purified lysate as
ABP-PP-BD04250 250 rxns PCR template.
*All Blood DNA Isolation & PCR Kits contain 2X
PCR Mastermix, also sold separately:
Cat #: ABP-PP-MM02961
♦ Please see reverse side
for Recommended PCR
Protocol & Troubleshooting
ProtocolsContinued Box 3 | Recommended PCR Setup

Allele-in One PCR 2X MasterMix 5µL

Whole Blood: Lysate 0.05 - 0.5µL**
Blood samples may be mixed with Isolation buffer without Forward Primer 0.2 - 0.5µL**
separation and removal of red cells. Mix 100 μL whole Reverse Primer 0.2 - 0.5µL**
blood (preserved or clotted) with 100 μL Isolation buffer, mix Fill to 10µL with nuclease-free water
well by inversion of tubes, and incubate at 50-60°C for 15
minutes. Use the resulting lysate for PCR reactions. \
Lysate Purification Protocol: **Lysate and primer concentrations vary based on
Important Reminders primer pair.

♦Add ethanol to AW buffer, wash buffer.

♦All centrifugation steps should be performed at ~14k rpm.

1. Add an equal volume of AB buffer to the Lysate.

2. Check that the Allele PG column is placed inside 2 Box 4 | Purification Kit Contents
mL collection tube.
Component 100 rxns 250 rxns
3. Transfer Lysate/AB mix to the Allele PG column.
Allele PG Column 100 250
4. Centrifuge for 1 minute and discard flow-through
2 mL collection tubes 100 250
inside collection tube.

5. Wash DNA by adding 700 μL AW buffer. 1.5 mL elution recovery 100 250
tubes
6. Centrifuge for 1 minute and discard flow-through
AB: Binding buffer 35 mL 87.5 mL
collected inside the collection tube.

7. Add an additional 500 μL AW buffer and centrifuge AW: Washing buffer* 12.5 mL 31.25 mL
for 1 minute. TE: TE buffer 10 mL 35 mL
8. Transfer Allele PG column to clean microcentrifuge
tube.
*Note: All volumes for AW (washing buffer) indicate volumes
9. Elute DNA by adding 30-50 μL TE buffer directly to
before the addition of 95-100% ethanol. For any (100)
membrane of Allele PG column. reaction/prep kit add 50 mL, and for any (250) reaction/ prep
kit add 125 mL 95-100% ethanol.
10. Incubate at room temperature for 1 minute and
centrifuge for 1 minute.

11. Store DNA at -20°C.

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Allele Biotech-Introducing Cost Effectiveness to Research