Drug Transporter Detection Kit Using 25-Multiplex PCR Assay
Jinsung SMR Co., Ltd
http://www.jssmr.co.kr
E-mail: js0228@chol.com
90-39, Bangrim-dong, Nam-gu Gwangju 503-050, c
Republic of Korea
Phone:+82-62-672-7631
Fax:+82-62-672-7632
Headquarters
Seoul Office
49-3,Munpyeong-dong,
Daedeok-gu,Daejeon306-220,
Korea
Phone:+82-42-936-8500
Fax:+82-42-930-8600
Bioneer Corporation
http://www.bioneer.co.kr
USA Bioneer, Inc.
412, 1580, DMC Hi-tech industry
center, Sangam-dong, Mapo-gu,
Seoul, 121-835, Republic of Korea
Phone:+82-2-598-1094
Fax:+82-2-598-1096
Order
1000 Atlantic Avenue, Alameda, http://www.jssmr.co.kr
CA 94501 USA
E-mail:js0228@chol.com
Toll free : 1-877-264-4300
Fax:+82-62-672-7632
Fax : 1-510-865-0350
E-mail:order.usa@bioneer.com
I. Introduction
We provides scientists with 25plex PCR assay kits to simultaneously amplify 5 sets of 5 genes, providing the rapid detection of mRNA expression for drug
transporter genes, including 24 drug transporter genes and one housekeeping gene; 17 ABC transporters (ABCA2, ABCA3, ABCB1, ABCB4, ABCB5, ABCC1,
ABCC2, ABCC3, ABCC4, ABCC5, ABCC6, ABCC9, ABCC10, ABCC11, ABCG1, ABCG2 and CFTR), one non-ABC transporter (MVP), 4 SLC transporters
(SLC28A1, SLC28A2, SLC29A1, SLC29A2), 2 copper transporters (SLC31A1 and ATP7B) and one house-keeping gene (GAPDH).
Drug Transporter Detection Kit Using 25-Multiplex PCR Assay was designed by Chemical-Mediated Hotstart method. The DNA polymerase is inhibited by the
pyrophospate, but activated upon pyrophosphate hydrolysis by the thermostable pyrophosphatase (Patent pending). This prevents the formation of mis-primed
products and primer-dimers during the reaction set up process resulting in improved PCR specificity.
In addition, Drug Transporter Detection Kit Using 25-Multiplex PCR Assay makes hot-start PCR simple and easy, eliminating the extra handling steps and
contamination risks associated with conventional hot-start methods.
Notice to Purchaser
This enzyme is specifically optimized for increasing base incorporation rate by inactivating 5’->3’ exonuclease activity. Therefore, this is not recommended to use
for Real Time PCR using Taqman® probe, which will be released soon.
II. Product Components
Description
Number of Reactions
Drug Transporter Detection Kit Using
25-Multiplex PCR Assay
JS-001 a
32 reactions x 5 sets
aThe
total Mg2+ concentration presented in the final 1 x working solution is 1.5 mM.
The total dNTP concentration presented in the final 1 x working solution is 1 mM (250 uM of each dNTP).
III. PCR Protocol using Drug Transporter Detection Kit Using 25-Multiplex PCR Assay
This protocol serves only as a guideline for PCR amplification. Optimal reaction conditions such as incubation times, temperatures, and amount of template DNA
may vary and must be individually determined.
Notes:
• Each PCR program be sufficient to started an Pre-denaturation step of 5 min at 94°C.
• Set up reaction mixtures in an area separate from that used for DNA preparation or PCR product analysis.
• Use disposable tips containing hydrophobic filters to minimize cross-contamination.
1.
Prepare the Drug Transporter Detection Kit Using 25-Multiplex PCR Assay and template cDNA.
2.
Add template DNA (≤1,000ng/reaction) to the individual PCR tubes.
A negative control (without template DNA) should always be included. It is not necessary to keep PCR tubes on ice as nonspecific DNA synthesis cannot
occur at room temperature due to the inactive state of Hot Start DNA Polymerase.
Component
Volume/reaction
Drug Transporter Detection Kit Using
25-Multiplex PCR Assay
20ul
Final concentration
1 unit of HotStart DNA Polymerase
1x PCR Buffer*
250 µM of each dNTP
Template DNA
Template DNA, added in step 2
Total volume
Variable
200 ~ 1,000 ng/reaction
20 µl
–
* Contains 15 mM MgCl2
3.
Add distilled water to Drug Transporter Detection Kit Using 25-Multiplex PCR Assay tubes until the total volume of mixture becomes 20 l.
Dissolve the lyophilized blue pellet by vortexing and spin-down.
4.
When using a thermal cycler with a heated lid, do not use mineral oil. Proceed directly to step 5. Otherwise, overlay with approximately 50 µl of
mineral oil.
V109C3
Blazing the Biotechnology Trail
Drug Transporter Detection Kit Using 25-Multiplex PCR Assay
Jinsung SMR Co., Ltd
http://www.jssmr.co.kr
E-mail: js0228@chol.com
90-39, Bangrim-dong, Nam-gu Gwangju 503-050, c
Republic of Korea
Phone:+82-62-672-7631
Fax:+82-62-672-7632
5.
Headquarters
Seoul Office
49-3,Munpyeong-dong,
Daedeok-gu,Daejeon306-220,
Korea
Phone:+82-42-936-8500
Fax:+82-42-930-8600
Bioneer Corporation
http://www.bioneer.co.kr
USA Bioneer, Inc.
412, 1580, DMC Hi-tech industry
center, Sangam-dong, Mapo-gu,
Seoul, 121-835, Republic of Korea
Phone:+82-2-598-1094
Fax:+82-2-598-1096
Order
1000 Atlantic Avenue, Alameda, http://www.jssmr.co.kr
CA 94501 USA
E-mail:js0228@chol.com
Toll free : 1-877-264-4300
Fax:+82-62-672-7632
Fax : 1-510-865-0350
E-mail:order.usa@bioneer.com
Program the thermal cycler according to the manufacturer’s instructions.
Each PCR program is equal to Pre-denaturation step at 94°C for 5 min. A typical PCR cycling program is outlined below.
Time
Pre-denaturation step:
3-step cycling
Temperature
5 min
94°C
Denaturation
30sec
94°C
Annealing
30sec
57°C
Extension
30sec
72°C
Number of cycles:
35
Final extension:
5 min
Additional comments
Equal to General PCR’s Initial activation step
72°C
6.
Place the PCR tubes in the thermal cycler and start the cycling program.
Note: After amplification, samples can be stored overnight at 2–8°C or at –20°C for longer storage.
7.
Load 5ul ~ 10ul of samples on agarose gel without adding loading dye mixture and perform 2.0% agarose gel electrophoresis.
Ordering Information
Tube type
Reaction
Cat.No.
Description
0.2 ml Tube
20 ul
JS-001
0.2 ml thin-wall 8-strip tubes with attached cap / 32 tubes x 5 sets
V109C3
Blazing the Biotechnology Trail