KT23 Whole Blood DNA Extraction Kit, GLPL01

Whole Blood DNA Extraction Kit GeNei™ Whole Blood DNA Extraction Kit GeNei™
Whole Blood
DNA Extraction Kit
Cat No. 2102300011730

Revision No.: 00050516
© Genei Laboratories Private Limited, 2016 © Genei Laboratories Private Limited, 2016
Notes:
CONTENTS
❖ Description 1
❖ Materials Supplied 4
❖ Protocol 1 6
❖ Protocol 2 8
❖ Trouble Shooting 10
❖ Ordering Information 12
❖ Flow Chart 14
15
Description: Flow Chart
DNA extraction kit from whole blood is designed to provide Pipette 300 µl of blood in EDTA coated 1.5 ml vials
quick, reliable and reproducibly higher yields of genomic DNA
from fresh blood as well as frozen blood. To avoid clotting,
blood should be collected in EDTA coated collection Lyse the RBCs by addition of 1 ml of 1X Solution A
vials/tubes. The first step in the extraction procedure is the
lysis of the red blood cells using Solution A, followed by lysis
of the white blood cells and their nuclei using chaotropic Spin at 8000 rpm for 5 min at room temperature
Solution B. Finally, genomic DNA is concentrated and
desalted by alcohol precipitation. The kit ensures extraction of Repeat the lysis until a clear white pellet of WBC is seen
highly pure DNA that can be used directly for PCR
amplification and restriction digestion.
To the WBC pellet, add 600 µl of Solution B
Highlights: and mix gently for clear lysis
• No binding resin – avoids shearing.
• No RNase treatment
• Does not involve phenol extractions. Centrifuge at 10,000 rpm for 10 min at room temperature

• Quick protocol
• Recovery from 300 µl of fresh Human Blood (8– 10 µg) Collect supernatant, add 0.9 ml absolute ethanol, mix
• Recovery from 300 µl Frozen Human Blood (3 – 5 µg) and centrifuge at 12000/10000 rpm for 25 minutes at 4°C.
Applications:
Wash the precipitated DNA twice with 0.5 ml of 75% ethanol
Purified DNA can be used for downstream applications like
PCR, Restriction Digestion.
Centrifuge at 10000 rpm for 5 minutes at 4°C
Time Required:
Total Time: 90-100 minutes
Air dry the pellet for 5 min add 50 -100 µl of Solution C
Incubate the vial at 55ºC for 5 –10 mins or leave at 4°C

for 24-48 hrs
Centrifuge at 10000rpm for 2 min.
Store the supernatant (DNA) at -20°C

1 14
M a b a b a b a b
Notes:
Lane 1 : -20C
Day 0 Day 3 Day 5 Day 10
M: /Hind III digest

Lane 2 : 4C
Lane 3 : RT
Fig 1: Analysis of Human genomic DNA purified using Whole Blood Genomic
DNA Extraction kit from blood samples (collected using EDTA as the
anticoagulant), stored at 4C, -20C and room temperature (RT) for 10 days, on
1% agarose gel.
M a b c a b c a b c
Day 0 Day 3 Day 10
Fig 2: Analysis of PCR amplified product of Human genomic DNA purified using
Whole Blood Genomic DNA Extraction kit from blood samples stored at 4°C
upto 10 days.
M: PCR Medium Range Marker

a: DNA Amplified using Primers for Abelson Murine Leukemia Viral Oncogene
(ABL)
b: DNA Amplified using Primers for Porphobilinogen Deaminase (PBGD)
c: DNA Amplified using Primers for β2-Microglobulin (βMG)
2
13
Ordering Information:
M a b c a b c a b c
Product Size Cat No.
Day 0 Day 3 Day 10
M: PCR Medium Range Marker
a: DNA Amplified using Primers for ABL
Whole Blood 1 EA 612102300011730
b: DNA Amplified using Primers for PBGD DNA Extraction Kit
c: DNA Amplified using Primers for βMG (Consumables for 50 Preparations)
Fig 3: Human genomic DNA purified using Whole Blood Genomic DNA Other Related Products:
Extraction kit from blood samples stored at -20°C upto 10 days, assessed for its Cat No. Related Products
performance in PCR with 3 different primers
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M a b c a b c a b c High Concentration Restriction Enzymes

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M: PCR Medium Range Marker 2115400021730 GeneiPureTM Gel Extraction Kit
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a: DNA amplified using Primers for ABL 2115900031730 GeneiPureTM Bacterial DNA Purification Kit
b: DNA amplified using Primers for PBGD
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Purification Kit
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Fig 4: Human genomic DNA purified using Whole Blood Genomic DNA
Email:
Extraction kit from blood samples stored at RT upto 10 days, assessed for its Sales: sales@geneilabs.com
performance in PCR with 3 different primers
Customer Support:
techsupport@geneilabs.com
3 12
Possible Cause Suggestions Materials Supplied:
Rehydration of Do not air dry the pellet for longer The list below provides information about the materials
DNA period as this would reduce the supplied in the kit. The components should be stored as
solubility of DNA. suggested.
Increase the solubility of DNA by Quantity

keeping the DNA at 4ºC for Materials Store
24–48 hours after addition of 2102300011730
Solution C (50 reactions)
Solution A 100 ml 4ºC
Solution B 35 ml 4ºC
Depending upon the DNA pellet
size (recovered after precipitation) Solution C 10 ml 4ºC
add Solution C in the range of
50 µl – 150 µl along the sides of the Materials required but not supplied:
vial very gently. Equipment : Microcentrifuge, Dry Bath
Reagent : EDTA coated blood collection tubes, Distilled
Ethanol & Ice-cold distilled water (Sterile)
Handle the DNA sample very gently
Other Requirements: 1.5 ml vials (Sterile),
to avoid shearing
15 ml Collection Tubes
Improper Reagent Dilute 3 X Solution A to 1 X before
preparation starting the experiment
DNA diffuses Increase amount of glycerol in Gel
while loading on Loading Buffer used to load DNA on
the agarose gel the agarose gel.
4
11
Note: Trouble Shooting:
• Read the entire procedure carefully prior to Observation: Low or no DNA recovery (Smear or band
starting the experiment. intensity low or not seen)
• When handling blood samples follow recommended
protocols for bio hazardous materials as applicable in Possible Cause Suggestions
your institution. Clotted blood used Avoid clotting of blood by using
• Avoid contamination of the reagents by using fresh tips anti coagulants like 50 µl (50 mM)
every time. EDTA or 5-10 units of heparin for
• While handling Solution B, wear gloves as it is 300 µl of blood
corrosive in nature Storage of Blood Good yields are seen with fresh
• Solution A is supplied as 3X concentrate. Dilute the sample blood or blood stored at 4°C for
required amount with sterile ice cold distilled water to less than 1 week.
1X concentration (i.e for every 1 ml of Solution A add
2 ml of distilled water) before use. Do not store the Yield from frozen blood is
diluted Solution A comparatively lower.
• Add 50 µl of 50 mM EDTA (anti coagulant) to 1.5 ml Incomplete Lysis Do not keep the sample in
vials prior to collecting 300 µl blood. Solution A for longer periods as it
• For the scale up of the protocol check the volumes would lyse the nucleated cells. The
stated on page 8. total time should not exceed
• Prepare 75% Ethanol by diluting the distilled 30 minutes.
100% ethanol with sterile distilled water.
Look for clear lysis after addition of
• Removal of Red Blood Cells (RBC) is critical step.
Solution B .Mix by investing the
Lysis of RBC will vary from sample to sample and may
tube or resuspend the pellet by
require 1 to 6 washes with 1X Solution A. Solution A is
gently pipetting up and down.
provided for 6 washes per sample.
DNA precipitation Do not spool the DNA as the
• For optimum recovery of DNA preferably use fresh
volume of the sample taken ie 300
blood which has been stored for not more than a day,
µl of blood is low and inturn the
as stored blood shows decreased recovery of DNA
DNA recovery will be low.
(refer figure 1).
• On prolonged storage of blood samples, degradation
of DNA is observed, which affects the performance of Use ethanol for precipitation of
the DNA in PCR (refer figures 2-4). DNA.
5
10
DNA concentration: Protocol 1:

To determine the DNA concentration, remove an aliquot, For DNA Extraction from Fresh Blood or blood stored at
dilute with water or TE (10 mM Tris-Cl, 1 mM EDTA, pH 8.0) Room Temperature/4°C.
and measure absorbance at A260. For double stranded DNA, 1. Collect desired amount of blood in EDTA coated 15 ml
1 OD at A260 approximately equals 50 µg/ml. blood collection tubes/vials. Store at 4°C.
Note: Do not store for more than 7 days.
Amplification of DNA by PCR: Add 0.1 to 1 g of DNA to 2. Pipette 300 l of blood in a EDTA-coated 1.5 ml vial.
PCR mix and follow the PCR protocol. Centrifuge at 5000 rpm for 8 minutes at room
temperature.
Digestion of DNA with Restriction Enzymes: Allow the
digestion to continue for 4 to 24 hours under optimal 3. Pipette out the serum ie the supernatant.
conditions. Usually, 80 to 90% of DNA is digested if 7-10 units 4. Resuspend the pellet in 1 ml of 1X Solution A (ice-cold).
of enzyme is used per g of DNA Mix by inverting the vial, leave at room temperature for
5-7 minutes, mixing intermittently about 2-4 times.
Centrifuge at 8000 rpm for 5 minutes at room
temperature.
5. Remove the supernatant (lysed RBCs) by pipetting.
Immediately resuspend the pellet in 1 ml of Solution A and
centrifuge at 8000 rpm for 5 minutes at room temperature.
6. Carefully pipette out the lysed RBCs i.e. supernatant (if
seen) above the White Blood Cells (WBC). Do not disturb
WBC which appear as a tiny white pellet.
Note:
• Observe for RBC lysis after addition of
1X Solution A. If red cell debris is still present
along with the white nucleated cell pellet, repeat
Step 4.
• Do not keep the sample in Solution A for longer
periods as it would lyse the nucleated cells as
well. The total time should not exceed 30 minutes
7. To the WBC pellet add 600 l of Solution B, resuspend
well and leave at room temperature for 5 minutes for
complete lysis.
9 6
8. Centrifuge at 10,000 rpm for 10 minutes at room 3. At the time of extraction, remove the frozen blood sample
temperature. (i.e. pellet from step 2) from the freezer and thaw at room
9. Collect the supernatant in a fresh 1.5 ml vial (Sterile). temperature.
10. To this add 0.9 ml of distilled absolute ethanol, mix gently 4. Resuspend the pellet in 1 ml of 1X Solution A (ice-cold).
by inverting the vial. Mix by inverting the vial. Leave at room temperature for
11. Centrifuge at 10,000 rpm for 25 minutes at 4°C. Discard 5-7 minutes. Mix intermittently about 2-4 times. Centrifuge
the supernatant. at 8000 rpm for 5 minutes at room temperature.
Note: 5. Remove the supernatant by pipetting (lysed RBCs).
• Strands of DNA precipitates out if the WBC Immediately resuspend the pellet in 1 ml of Solution A and
pellet is more. centrifuge at 8000 rpm for 5 minutes at room temperature.
• Do not spool DNA. 6. Repeat Step 6, 3-4 times, until a white WBC pellet is
12. Wash DNA twice with 0.5 ml of 75% ethanol by giving seen.
short spins at 10000 rpm for 5 minutes at 4°C. 7. Carefully pipette out the lysed RBC i.e. supernatant (if
13. Discard the supernatant and air-dry the DNA pellet for 5 seen) around the WBC after final centrifugation.
minutes only at room temperature. Note: Do not disturb WBCs which appear as a tiny
Note : Do not overdry as it affects solubilisation of white pellet.
DNA in Solution C. 8. Proceed with step 7 of Protocol 1 for DNA.
14. To the pellet add 50-100 µl of Solution C.
15. Incubate at 55°C for 10-15 minutes to improve the
solubility. Note:
Note: To improve recovery, leave in Solution C at 4°C For Scale up of the protocol refer to the table below:
for 24-48 hrs.
16. Centrifuge at 10,000 rpm for 2 minutes to remove any 0.3 ml 1 ml 2 ml
insoluble material. Solution A 1 ml 3.5 ml 1 ml
17. Collect the supernatant, which contains DNA. This DNA Solution B 0.6 ml 0.6-0.8 ml 1.2-1.5 ml
can be used immediately or can be stored at –20° C for Solution C 50-100 µl 100-200 µl 100-200 µl
future use.
Protocol 2
For DNA Extraction from Frozen Blood
1. Collect blood in EDTA coated blood collection tubes/vials
as in Protocol 1.
2. Pipette 300 µl of blood in a 1.5 ml vial and centrifuge at
5000 rpm for 5 minutes at room temperature. Pipette out
the serum i.e. the supernatant.
Note: Pellet obtained can be frozen at –20°C.
7 8

KT23 Whole Blood DNA Extraction Kit, GLPL01

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Whole Blood DNA Extraction Kit GeNei™ Whole Blood DNA Extraction Kit GeNei™

Cat No. 2102300011730

Incubate the vial at 55ºC for 5 –10 mins or leave at 4°C

Centrifuge at 10000rpm for 2 min.

Store the supernatant (DNA) at -20°C

Day 0 Day 3 Day 5 Day 10

M: /Hind III digest

Day 0 Day 3 Day 5 Day 10

Day 0 Day 3 Day 5 Day 10

Day 0 Day 3 Day 10

M: PCR Medium Range Marker

M a b c a b c a b c High Concentration Restriction Enzymes

Increase the solubility of DNA by Quantity

DNA concentration: Protocol 1:

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