Download Genotyping of knock-out mice | Nippon

Application Note 2013〈16〉
Customer feedback on products
Product Name
Manufacturer
Application
： KAPA MG Kit (KK7150MG)
KAPATaqEXtra HotStart Readymix with Dye (KK 3606)
： KAPA BIOSYSTEMS
： Genotyping of knock-out mice
The following data were provided by the courtesy of Dr. Mamoru Aoto of Department of Circulatory Physiology, Graduate School of
Medicine, Ehime University, Japan.
Experimental conditions
①Conventional method (Manufacturer T's product)
＜Extraction of mouse tail DNA＞
Mouse tail（2mm） in 1.5ml tube
↓
+ 75µl Alkaline Lysis Buffer
(25mM NaOH, 0.2mM EDTA)
↓
95℃, 30min
↓
4℃
↓
+ 75µl Neutralization Buffer
(40mM TrisHCl, pH 5)
Vortex well
＜PCR reaction mixture composition＞
10× Reaction buffer
2.5µl
2µl
2.5mM MgCl2
2.5mM dNTP mix
2.5µl
10µM Fwd Primer
1.25µl
10µM Rev Primer
1.25µl
Template
1µl
Manufacturer T's enzyme
0.25µl
MilliQ water
14.25µl
total
25µl
②KAPA MG Kit
＜Extraction of mouse tail DNA＞
Followed the instructions attached to the kit.
③KAPATaq EXtra HotStart ReadyMix with dye (2×)
＜Extraction of mouse tail DNA＞
Same as the conventional method.
＜PCR reaction mixture composition＞
KAPA2G Robust
HotStart ReadyMix (2x)
12.5 µl
10µM Fwd Primer
1.25µl
10µM Rev Primer
1.25µl
Template
1µl
MilliQ water
9µl
total
25µl
＜PCR reaction mixture composition＞
KAPATaq EXtra
HotStart ReadyMix with dye (2x)
12.5 µl
10µM Fwd Primer
1.25µl
10µM Rev Primer
1.25µl
Template
1µl
MilliQ water
9µl
total
25µl
● PCR device：BIO-RAD T100 Thermal Cycler
● PCR program
95 ℃,
3min ×1 cycle
95 ℃, 15sec
64 ℃, 15sec ×35 cycle
72 ℃, 15sec
16 ℃,
∞
● Target amplicon size：163bp
Results
KAPA Conventional
method
MG Kit Manufacturer T
＋＋−−＋＋−−
KAPATaq Conventional
method
EXtra Manufacturer T
＋＋−−＋＋−−
The operation procedures and PCR results were compared among two KAPA kits (KAPA MG
Kit, KAPA TaqEXtra HotStart ReadyMix with dye) and a conventional method (Manufacturer T).
Summary of comparison between KAPA kits and the conventional method (Manufacturer T)
・Neither of the kits detected any non-specific bands that were detected in the conventional
method.
・Both kits were equivalent to the conventional method in terms of amplification efficiency.
・Both kits support easy preparation of the PCR reaction mixture and therefore prevent
mistakes (particularly for beginners).
・KAPA MG Kit requires shorter time for DNA extraction compared to the conventional
method.
・KAPA TaqEXtra contains a dye, so electrophoresis can be performed directly after the PCR.
M
: Marker
1, 2 : Knock-out mice (+)
3, 4 : Wild-type mice (−)
Electrophoresis conditions
1% Agarose gel, 100V, 30 min
Reaction mixtures were applied in 10 μl aliquots.
The marker was applied in 5 μl aliquot.
<Customer's comments>
We have been detecting KO alleles by PCR for genotyping.
Both of the Kapa Biosystems kits suppressed non-specific amplification and produced easy-to-comprehend results.
Inexperienced students (medical school freshmen) could successfully perform genotyping without making any mistakes.
Nippon Genetics Co.,Ltd http://www.n-genetics.com
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KP2013AUG7150MG