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TB-MAC Detection Kit hand Book
The TB-MAC detection Kit is a research reagent intended for detection of Mycobacterium
Tuberuculosis complex and M. avium complex (MAC) genome .
This product uses DNA chromatography strip : C-Pas ( GB6), which is produced by TBA , co., Ltd . Patent -Licences by NGK insulators, Ltd.
After PCR , we can visually judge the existence of the target gene with DNA Chromatography method.
This kit is optimized for 10μl of PCR system .
Target : Detection Line
Internarl control : Tag 6
MAC Tag 4
(M.avium, M.Intracellular, M.Chimera,M.Genavense. Tag 4
M.Lentiiflavum, M.intermedium, M.arosiense, Tag 4
M.Colombiense,M.Florentinum, M.mantenii Tag 4
M.Stomatepiae,M.Vulneris , M.youngonense Tag 4
M.tuberculosis complex Tag2
(M.tuberuculosis, M. Vulneris , M . Youngone nse) Tag2
Low-Profile PCR tube
4.Other Necessary things
- Premix PCR Enzyme
- Recommendation : SYBR Premix Ex Taq ) Tli RNaseH plus)(Takara RR20)
- Quick TaQHS
- Gene amplification machine .
- Recommendation:Quick Bath ) Thermo Gen QM-0104A)
- Vortex Mixer
TB-MAC detection Kit Hand Book
5-1.Preparation of DNA solution
Please extract DNA from Samples .
5-2. Gene amplification by PCR
1)Prepare reaction mixture.
Add the following premix PCR solution to PCR tubes.
Premix PCR solution 5.0 μl
Primer Solution 2.5 μl
Total Volume 7.5 μl
2)Add 205 μl of sample DNA solution ( Total 10 μl).
3)Amplify nucleic acids by PCR.
Be sure to close the PCR caps to avoid liquid leakage and evaporation during PCR. Then, Perform PCR under the following condition using Quick Bath.
PCR condition ( prese program No. SS65)
97°Ｃ 2 Min
97°Ｃ 5 sec
65 °Ｃ 10 sec
5-4.Notices in use of DNA chromatography strips : C-PAS(GB6)
Be sure to hold a part of absorbing pad of C-PAS) GB6) with dried hands in use .
Touching the parts other than absorbing pad and holding with me hands may result in and insufficient reaction.
TB-MAC detection Kit Hand Book
- Absorbing Pad
Be sure to hold a part of absorbing pad of C-PAS(GB6) in use .
- Position Marker
The position of emerging lines is discriminated by these lines.Position makers are always appeared with out without reaction.
- Detection Line
Blue positive lines that exist before reaction temporary disappear when the solution passes through.
After that , reaction line appears again depending on the reaction .
Blue lines before use are gradually faded , but these changes don affect the reaction .
5-4.Reaction Using DNA chromatography Method
1）Take out Latex solution and Dilution buffer from refrigerator and keep at room temperature.
2)Prepare the Latex-Working solution by dilution the Latex Solution 1:10 in dilution Buffer.
Mix the solution uniformly with vortex mixer by.
3)Open the caps of PCR tubes after PCR and add 11 μl of the latex working solution . Mix the solution uniformly by pipetting.
4)Insert C-PAS (GB6)in tube of step “3)”.Top opposite of absorbing pad must be inserted tubes.
5)Leave them at room temperature for 10 minutes for developing . Perform the reaction at room temperature (20 ～30°Ｃ）.
A low temperature causes flash – positive detentions.
5-5.Judgement of the results
Negative : When no target gene exists in sample , the blue line at Tag 6(IC) line appears.
Positive :When target gene exists in sample , the blue line at detection line appears.
IC line Often becomes faint when target gene is amplified.