Manufacturing of molecular reagents and services in Egypt

ABOUT US
Applied Biotechnology is manufacturing company for
molecular biology reagents. Also, Applied Biotechnology
company offers many molecular services to the scientific
community. The company mission is offering good quality
molecular reagents and services at reasonable prices to help
the researchers. The company operates by well trained
researcher. Also, we try to offer professional customer
support to our customers. The company has well equipped
molecular diagnostic laboratory. The company headquater is
located in Ismailia province, Egypt.

Our Adress is Eleshreny Street beside Tiba Pastry, Ismailia,

Egypt. you can contact us by sending message to our
facebook page https://www.facebook.com/appbiotech/
Molecular
Regents
manufacturer
Molecular
Diagnostic
services

Molecular
research
services

Products and Services

Nucleic Acid Extraction
DNA Mini extraction kit (spin Column)

6-8

Total RNA Mini extraction kit (spin Column)

9-10

PCR and RT-PCR

2X Red Master mix

11

2X Taqman Master mix

12

2X SYBR green Master mix

13

cDNA synthesis Kit

14

Electrophoresis
100 bp DNA ladder

15

100 bp plus DNA ladder

16

Molecular research Services

17

DNA extraction
RNA extraction
Polymerase chain reaction
Gel electrophoresis
Real time PCR
Bacterial and fungal identification
Gene Sequencing

Molecular Diagnostic Services

18

Viral Diseases Molecular Diagnostics

Rotavirus group A (RVA) Taqman real time PCR
West Nile fever virus Taqman real time PCR
Pan-Herpes virus PCR
Pan-Coronavirus PCR
Rift Valley Fever virus SYBR Green Real time PCR
Influenza Virus group A Taqman real time PCR
Foot and mouth disease virus Taqman real time PCR
Foot and mouth disease virus serotyping
Bovine coronavirus Taqman real time PCR
Bovine respiratory syncytial virus Taqman real time PCR
Bovine Viral diarrhea virus Taqman real time PCR
Infectious bovine rhinotracitis (IBR) Taqman real time PCR
Boivne Ephemeral Fever Taqman real time PCR
Capripox Taqman real time PCR
Peste des petitis ruminants Taqman real time PCR
Canine distmper Taqman real time PCR
Canine parvovirus Taqman real time PCR
Newcastle disease virus Taqman real time PCR
Infectious bronchitis Taqman real time PCR

Bacterial Diseases Molecular Diagnostics

Brucella Taqman real time PCR
Leptospira Taqman real time PCR
Mycoplasma Bovis Taqman real time PCR
Pan-bacteria 16srRNA PCR
Pan-Rickettsia PCR
Anaplsma Marginalis PCR
Anaplasma Phagocytophilum PCR

Parasitic Diseases Molecular Diagnostics

Pan-Nematodes PCR
Pan-Trypanosoma PCR
Pan-Piroplasm PCR
Trypanosma evansi Taqman real time PCR
Tritrichomonas Fetus Taqman real time PCR

Nucleic Acids Extraction

DNA Mini extraction kit (spin Column)

. Kit Content, Storage Condition and Stability

Content
DNA lysis Buffer
Proteinase K (20mg/ml)
Washing Buffer 1 (WB1)
Washing Buffer 2 (WB2)
Spin columns
Elution buffer

Storage
RT
-20C
RT

RT
RT
RT

50 preps
12 ml
1 ml
10.5 ml
6 ml
50
12ml

All reagents, when stored properly, are stable for 24 months.

Notes:
1. Please add absolute ethanol to WB1 and WB2 before first use. Mix well and mark the check box
labeled on the bottles to indicate that the ethanol has been added.
2. Please ensure the bottles tightly capped when not in use, prevent reagents from evaporating,
oxidation and pH change.

II. Principles:
The kit has a unique lysis buffer and Protease K to rapidly lyse cell and inactivate cellular nuclease,
then the DNA selectively adsorbs to silicified membrane in high salt solution. Cellular metabolite,
proteins and inhibitors are removed by serial washing by potent two washing buffers. Finally the
purified DNA eluted from silica membrane by low salt elution buffer.

III. Features
1. No need of harmful phenol and chloroform.
2. Simple and rapid. One preparation can be completed in 20 min.
3. Multi-elution ensures high-purified DNA. The DNA yield achieving 3-6g in 100 ul.

IV. Samples preparation

Whole blood: Add 200 ul of fresh or freezed blood in 1.5 ml centrifuge tube.
Bacteria grow in broth: Add 200 ul of bacterial broth in 1.5 ml centrifuge tube or spin 1.5 ml of
bacterial broth in 1.5 ml tube, discard the supernatant and suspend the bacterial pellet in 200 ul
of saline.
Bacterial colonies: by bacteriological loop collect the bacterial colonies in 200ul of saline in 1.5
ml centrifuge tube
Tissues: transfer a small part of tissue in 1.5 ml centrifuge tube contain 200 ul saline or grinding
the tissue in a morter and pestle contain liquid nitrogen, transfer the tissue powder in 1.5 ml
centrifuge tube.
Feces: Suspend the fecal material in saline, put the fecal suspension in bench for 5 minutes,
transfer 200 ul of the supernatant into 1.5 ml centrifuge tube.

V. Procedures
1. Add 200 ul of DNA lysis buffer and 19 ul of proteinase K to the prepared samples in 1.5 ml
centrifuge tubes.
2. Mix vigorously by pipetting and vortex. Please, avoid any splash into the automatic pipette or
leakage from 1.5 ml centrifuge tubes to avoid cross contamination.
3. incubate at 65C for 20 minutes in blood and bacteria. The incubation period should increase
up to 3 hours in case of tissues to ensure proper lysis. N.B the length of incubation varies, if you
find that tissue samples are well homogenate and completely lysed so you can proceed.
4. Vigorous vortexing during incubation to ensure proper lysis.
5. In case of tissue samples or any other samples that is not completely lysis, spin the tube at
12000 rpm for 1 minutes, transfer the supernant into fresh 1.5 ml centrifuge tube. This step is
important to avoid clogging of the spin columns.
6. Add 100 ul of isopropanol or absolute ethanol to the lysate. Mix well by inversion for 4 times.

7. Transfer the lysate into Spin column. Then centrifuge at 12000 rpm for a minutes. Discard
the filterate in the collection tube. If the column clogged by climbs or any debris, please try to
remove the blocking materials from column by tips of automatic pipette and spin the column
again.
8. Add 300 ul of WB1 into the spin column. Spin at 12000 rpm for 30 seconds. Discard the
filterate.
9. Add 300 ul of WB2 into the spin column. Spin at 12000 rpm for 30 seconds. Discard the
filterate.
10. Spin the empty column at 12000 rpm for 1 minutes to get rid of any remaining washing
buffer.
11. Dicard the collection tube and transfer the spin columns into a new 1.5 ml centrifuge
tubes.
12. Add 50 ul of elution buffer into the center of the spin column. Incubate at room
temperature for 5 minutes. Centrifuge at 12000 rpm for 30 seconds.
13. Add another 50 ul of elution buffer into the spin column and repeat the step before.
14. Store the total 100 ul of highly pure DNA in 20C for further applications.

Nucleic Acids Extraction

Total RNA Mini Extraction Kit (Spin column)

. Kit Content, Storage Condition and Stability

Content

Storage

50 preps

4
RT

37 ml
15 ml
30 ml
20 ml
50
15 ml

RNA lysis buffer

70% ethanol
Inhibitor removal buffer
Washing buffer (WB)
Spin columns
Elution buffer

RT

RT
RT
RT

Notes:
1 Please add ration ethanol to Buffer RW and 70% ethanol before first use. Mix well and
mark the check box labeled on the bottles to indicate that the ethanol has been added.
2 Please ensure the bottles tightly capped when not in use, prevent reagents from
evaporating, oxidation and pH change.

II. Principles:
The kit has a unique RNA lysis buffer that rapidly lyse cell and inactivate cellular nuclease, then the
RNA selectively adsorbs to silicified membrane. Cellular metabolite, proteins and inhibitors are
removed by serial washing by potent washing buffers. Finally the purified RNA eluted from silica
membrane by low salt elution buffer.
III. Procedures:
Note: Add absolute ethanol to Buffer WB and 70% ethanol.
1 Homogenization:
a. Tissues: Please homogenize tissue in 700 ul of RNA lysis buffer.
b. Blood: Please add 200 ul of blood to 700 ul of RNA lysis buffer, mix well by pipetting.
Vortex vigorously.
C. Fecal matter: Suspend the fecal materials in saline, leave the sample for 5 minutes at
room temperature. Transfer 200 ul of supernatant into 1.5 ml tubes. Add 700 ul of RNA lysis
buffer. Mix well by pipetting. Vortex vigorously.

2 Incubate the homogenized samples for 5 minutes at room temeprature to permit the
complete dissociation of nucleoprotein complexes.
3 Add 0.2 ml of chloroform. Cap sample tubes securely. Vortex vigorously. Incubate them at
room temperature for 2 to 3 minutes.
4 Centrifuge at 12,000rpm for 10 minutes at room temperature.
5 Transfer the aqueous phase to a fresh tube. Add 700 ul of 70% ethanol. Mix by inversion
for 3 times. Transfer the mixture and precipitate to a Spin-column AC (placed in collection
tube). If the mixture is too much, apply the mixture in successive application to the same
Spin-column AC.
6 Centrifuge at 10,000 rpm for 30s at room temperature. Discard the flow through. Reuse
the Spin-column and the collection tube.
7 Add 500l inhibitor removal buffer to the center of Spin-column AC to remove the protein.
Centrifuge at 12,000 rpm for 45s. Discard the flow through.
8 Add 500l Buffer RW. Centrifuge at 12,000rpm for 30s. Discard the flow through.
9 Add 500l Buffer RW. Centrifuge at 12,000rpm for 30s. Discard the flow through.
10 Replace Spin-column AC to the collection tube and spin for 2min to remove the residual
fluid.
11 Place Spin-column AC to a 1.5ml RNase-free centrifuge tube. Apply 50l of elution buffer
to the center of the column. Place it at room temperature for 2min. Centrifuge at
12,000rpm for 1min. return the column to centrifuge tube and add additional 50 ul
elution buffer, spin again. You will have a total of 100 ul RNA.
12 Store the highly purified RNA at -80C until use.

10

PCR and RT-PCR

2X Red Master Mix
Component
Product
2X Red Master Mix

Amount
1.25 ml

Storage: Up to 2 year at -20.

Description: 2X Red Master Mix is a premix and ready to use solution, including DNA
polymerase, buffer, dNTP Mixture and loading dye. It is easy, simple, has low contamination
only add primers and template to the Mix. The product can be directly cloned in T-vector
because of A base at 3 end.
Reaction Setup
Component
2MasterMix
Template DNA
Upstream Primer (20M)
Downstream Primer (20M)
Sterile Water

25L reaction
volume
12.5 L
X L
1L
1L
Up to 25L

50 L reaction
volume
25 L
X L
1L
1L
Up to 50L

Thermal cycler condition

Cycle
1
30-35
1

Step
1
1
2
3

Temperature
95
95
50-60
72

Time
2-5min
30s
30s
1min/1kb

72

5 min

11

2X Taqman Master Mix (probe)

Component
Product
2X Taqman Master Mix

Amount
1.25 ml

Storage: Up to 2 year at -20.

Description: 2X Taqman Master Mix is a 2X concentration of premix reagent contains DNA
polymerase, dNTP, Unique buffers contain Mg salt and enhancer. This product provides a
newly developed buffer which provides superior specificity, increased amplification
efficiency and high sensitivity. This mix do not contain any Rox reference dye.
Reaction Setup
Component
2MasterMix
Template DNA
Upstream Primer (10M)
Downstream Primer (10M)
Probe (10M)
Sterile Water

25L reaction
volume
12.5 L
X L
1.5 L
1.5 L
0.5 L
Up to 25L

Thermal cycler condition

Cycle
1
45

Step
1
1
2
3

Temperature
95
95
50-60
72

12

Time
3 min
15 seconds
30 seconds
30 seconds

2X SYBR Mix

Component
Product
2X Taqman Master Mix

Amount
1.25 ml

Description:
2X SYBR Mix is a 2X concentrate of premix containing DNA polymerase, SYBR Green I, dNTP,
optimized buffer and stabilizer specially designed for real-time PCR with intercalator method.
This mix do not contain any Rox reference dye.
Reaction Setup
Component

25L reaction
volume
12.5 L
X L
1.5 L
1.5 L
Up to 25L

2MasterMix
Template DNA
Upstream Primer (10M)
Downstream Primer (10M)
Sterile Water
Thermal cycler condition
Cycle
1
45

Step
1
1
2
3

Temperature
95
95
50-60
72

13

Time
3 min
15 seconds
30 seconds
30 seconds

H minus cDNA synthesis kit

This Kit contains M-MLV (H minus), optimal reaction buffer, dNTP, RNase-free water
and oligo (dT)18. The product can be directly used in 2nd strand synthesis, hybridization, PCR
amplification, real-time PCR, etc.
Component:
10 reactions

Product
M-MLV Reverse
Transcriptase200U/l
5First-strand Buffer

5.5 l

dNTP Mixture (10 mM

each)
Oligo(dT)18 Primer (50M)

22 l

RNase-free H2O

750 l

60 l

15 l

Procedure
1Add the following components:
Template RNA
Primer

0.2-2g
Reverse primer
20 picomole
Or oligo (dt)18
1 ul
Or Random primer
1 ul
Nuclease free water
Up to
13.5 ul
2. If the RNA template is GC-rich or contains secondary structures, incubate at 65C for 5
min. Chill on ice, spin down and place the vial back on ice.
3. Add the following components into each sample:
5First-strand Buffer

4l

M-MLV Reverse Transcriptase (200U/l)

dNTPs

0.5 l
2 ul

3. For oligo(dT)18 or gene-specific primed cDNA synthesis, incubate for 60 min at 42C. For
random hexamer primed synthesis, incubate for 5 min at 25C followed by 60 min at 42C.
Note. For GC-rich RNA templates the reaction temperature can be increased up to 45C.
4. Terminate the reaction by heating at 70C for 5 min. The reverse transcription reaction
product can be directly used in PCR applications or stored at -20C.
14

Electrophoresis
100bp DNA Ladder

Contains 11 discrete DNA fragments ranging in size from 100bp to 1500bp. This marker is suitable
for sizing linear double-stranded DNA fragments. All bands (except 500bp) are supplied at
approximately 40-50 ng/5l. The 500bp are 100ng/5l as a reference band. This ladder is premixed with loading dye and is ready to use.
Concentration
Reference Band 100 ng/5l, Other Bands 40-50 ng/5l
Storage
The 100 bp ladder is stable for at least 3 months at 4C or room temperature. For long term
storage, store at -20C.
Recommended Loading
3-5 l/Lane.
Contentsbp

100, 200, 300, 400, 500, 600,700, 800, 900, 1,000, 1,500bp

15

100bp plus DNA Ladder

The 100bp Plus DNA Ladder contains 14 discrete DNA fragments ranging in size from 100bp to
5kb (100, 200, 300,400, 500, 600, 700, 800, 900, 1000, 1500, 2000, 3000, 5000bp). All bands
(except 500bp) are supplied at approximately 40-50 ng/5l. The 500bp are 100ng/5l as a
reference band. This ladder is pre-mixed with loading dye and is ready to use.
Concentration
Reference Band 100 ng/5l, Other Bands 40-50 ng/5l
Storage
The 100 bp ladder is stable for at least 3 months at 4C or room temperature. For long term
storage, store at -20C.
Recommended Loading
3-5 l/Lane.
Contentsbp
100, 200, 300, 400, 500, 600,700, 800, 900, 1,000, 1,500bp
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Molecular Research Services

Nucleic Acid extraction
Just visit our facility or ship us your samples and receive your highly purified DNA or RNA by
our innovative extraction kits. Our facility is well equipped by calibrated pipettes. Therefore,
we can offer extraction of large number of samples in short time. We accept samples from
any laboratory in Egypt. Just ship us by Middle East Courier Company. Also, we can offer
measurement of DNA purity and concentration by Nanodrop or visualizing the nucleic acid
by gel electrophoresis.
PCR and Real time PCR
Our facility has many thermal cycler machines and real time PCR machine. Therefore, any
researcher can rent the equipment or ask us to set up the PCR experiments.
Sequencing services
Our company offer professional sequencing service. We accept the non purified PCR product
at 40-50 ul reaction volume. Our results are fast. We can also offer sequence trimming with
extra fees.
Bacterial and fungal identification
We offer bacterial and fungal identification by 16srRNA and 18srRNA amplification and
sequencing. Just ship us your isolates and we will do all the experiments.

For further information, just send us a message.

17

Molecular Diagnostic Services

Our company offers many molecular diagnostic assays for human and animal diseases, We
have a well optimized diagnostic tests. Furthermore, we have the primers, probes and positive
controls. The list of diagnostic assays that we offer is:

Viral Diseases Molecular Diagnostics

Rotavirus group A (RVA) Taqman real time PCR , West Nile fever virus Taqman real time PCR ,
Pan-Herpes virus PCR, Pan-Coronavirus PCR, Rift Valley Fever virus SYBR Green Real time PCR ,
Influenza Virus group A Taqman real time PCR Foot and mouth disease virus Taqman real time
PCR, Foot and mouth disease virus serotyping , Bovine coronavirus Taqman real time PCR,
Bovine respiratory syncytial virus Taqman real time PCR, Bovine Viral diarrhea virus Taqman
real time PCR, Infectious bovine rhinotracitis (IBR) Taqman real time PCR, Boivne Ephemeral
Fever Taqman real time PCR, Capripox Taqman real time PCR, Peste des petitis ruminants
Taqman real time PCR, Canine distmper Taqman real time PCR, Canine parvovirus Taqman real
time PCR, Newcastle disease virus Taqman real time PCR, Infectious bronchitis Taqman real
time PCR.

Bacterial Diseases Molecular Diagnostics

Brucella Taqman real time PCR, Leptospira Taqman real time PCR, Mycoplasma
Bovis Taqman real time PCR, Pan-bacteria 16srRNA PCR, Pan-Rickettsia PCR
Anaplsma Marginalis PCR, Anaplasma Phagocytophilum PCR.

Parasitic Diseases Molecular Diagnostics

Pan-Nematodes PCR, Pan-Trypanosoma PCR, Pan-Piroplasm PCR, Trypanosma
evansi Taqman real time PCR, Tritrichomonas Fetus Taqman real time PCR.

For further information, just send us a message.

18