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For general laboratory use.
Please centrifuge briefly before opening (volume ≤2 ml).Shipping: shipped on gel packs
Storage Conditions: store at -20 °C
avoid freeze/thaw cyclesShelf Life: 12 months
Form: liquid
Description:
RT-LAMP Colorimetric Kit – lyo ready is an optimised formulation containing a visible pH depending dye for rapid and easy detection of the LAMP reaction.
This kit allows a fast, visual detection of the amplification by a clear pink-to-yellow color change.Lyophilisation
RT-LAMP Colorimetric Kit – lyo ready is recommended for preparation of lyophilised RT-LAMP assays.
The additive mix contains cryoprotectants and lyoprotectants for optimal stability and structure formation.Content:
Component Cap PCR-541S PCR-541L RT-LAMP Buffer yellow 800 μl 5 x 800 μl RT-LAMP
Enzyme Mixred 240 μl 5 x 240 μl RT-LAMP
Additive Mixviolet 1.6 ml 5 x 1.6 ml PCR-grade Water white 1.2 ml 3 x 1.2 ml Detection
The ongoing amplification can be observed directly with the naked eye by a color change of the assay. The color changes from pink to yellow as the amount of amplified DNA increases. Depending on the amount of template material used, the reaction is completed after 20 to 30 minutes.
The change depends on the pH change of the reaction system. Make sure not to inhibit this change by template DNA or RNA stored in Tris-buffer containing nucleic acid storage solution. PCR-grade Water is recommended to store RNA, DNA and primers.Assay design
Isothermal amplification is an extremely sensitive detection method and care should be taken to avoid contamination of setup areas and equipment with DNA of previous reactions. A common problem is amplification in no-template controls due to1. carry-over contamination or
2. amplification of unspecifically annealed primers or
primer dimer formations.As sensitivity and non-template amplification of in-silico designed primers may vary, the evaluation of 2-4 real primer sets before choosing a final set is recommended.
Assay set-up
Depending on the setup used, a reaction volume of 20-50 μl is recommended for most applications. Pipette with sterile filter tips and perform the setup in a separate area from the DNA preparation or analysis. No-template controls should be included with all amplifications.First, prepare a 10x conc. primer premix as follows:
primer stock conc. final conc. 100 μl 1 ml FIP 100 μM 16 μM 16 μl 160 μl BIP 100 μM 16 μM 16 μl 160 μl F3 100 μM 2 μM 2 μl 20 μl B3 100 μM 2 μM 2 μl 20 μl Loop F 100 μM 4 μM 4 μl 40 μl Loop B 100 μM 4 μM 4 μl 40 μl PCR-grade Water 56 μl 560 μl Set up for a liquid isothermal amplification assay
Prepare a master mix as follows:
component stock conc. final conc. 20 μl 50 μl RT-LAMP Buffer 5 x 1 x 4 μl 10 μl RT-LAMP Enzyme Mix 18 x 1 x 1.1 μl 2.8 μl Primer Mix 10 x 1 x 2 μl 5 μl Template RNA <100 ng/assay x μl x μl PCR-grade Water fill up to 20 μl fill up to 50 μl Set up for a lyo-ready isothermal amplification assay for freeze-drying
Prepare a master mix as follows:
component stock conc. final conc. 20 μl 50 μl RT-LAMP Buffer 5 x 1 x 4 μl 10 μl RT-LAMP Enzyme Mix 18 x 1 x 1.1 μl 2.8 μl Primer Mix1 10 x 1 x 2 μl 5 μl RT-LAMP Additive Mix 2.5 x 1 x 8 μl 20 μl PCR-grade Water fill up to 20 μl fill up to 50 μl 1 optional, if primers should be included in the lyophilised mix
Dispensing the master mix:
Vortex the mix prepared above thoroughly to assure homogeneity. Dispense the appropriate volume to each PCR tube or well of the plate.Lyophilisation of the master mix:
Use a freeze dryer or sublimator to freeze-dry the prepared master mix. Follow the instructions provided by the freeze-dryer manufacturer.Final assay preparation:
To prepare the final liquid assay, rehydrate the lyophilisate with primers (if not already included in the lyophilisate) and template RNA to obtain the final assay volume. Do not exceed 100 ng RNA per assay. Add PCR-grade Water for no-template controls. Close or seal the tubes/plates and centrifuge briefly to remove bubbles.Amplification and detection
- Use a thermoblock or PCR cycler to run the assays
- Set the instrument to a constant incubation temperature between 60 - 65°C (depending on the primer annealing temperature)
- Check the visible color change in appropriate intervals for up to 30 min.
Trouble shooting
If amplification in no-template controls occurs the following points should be reviewed.The pink-to-yellow color change depends on the pH change of the reaction system. Make sure not to inhibit this change by template RNA stored in Tris-buffer containing nucleic acid storage solution. PCR-grade Water is recommended to store RNA, DNA and primers.
When using the product as a liquid mix (non‑lyophilized), the initial color may appear dark orange rather than pink. This is acceptable and does not indicate reduced performance; the expected color shift will still proceed towards yellow.
Cross contamination from environments
- Clean equipment and areas with "RNA/DNA Away” solution
- Replace reagent stocks and pre-mixes with new components
- Stop reactions at an earlier point of time before non-template amplification occur
Carry-over contamination from previous reaction products
- Avoid opening reaction vessels after amplification
- Use separate preparation area and equipment if post-reaction processing is necessary
Non-template amplification from primers
- Increase incubation temperature stepwise by 1-2 °C
- Design a new set of primers for the target sequence
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产品组分
组分内容
型号 规格 储存温度
RT-LAMP比色冻干即用型试剂盒
CNPCR541-01 200次 -20°C RT-LAMP比色冻干即用型试剂盒
CNPCR541-02 1000次 -20°C 操作手册
1 1 室温
*注意：在使用之前，应将溶液离心至管底。
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注意事项
保存建议 推荐蓝冰或冰袋运输。当您收到产品后，按照说明书建议保存各组分。 警告 本品仅供科研使用，请勿用于临床与诊断。 -
FAQ
1. RT-PCR反应后，无PCR产物或出现Smear，怎么办？
此时请从提取的RNA样品的纯度和添加量、基因的表达丰度、基因的长度和GC含量、引物的设计情况、参考文献的可信度以及RT-PCR条件的设定等方面加以考虑。
2. PCR反应时，反转录反应液的使用量使用多少较为合适？
反转录反应液的使用量可控制在1 μl～10 μl的范围。
3. 3′RACE PCR扩增产物经电泳分析后，有时为什么出现多条带现象？
可能是因为未知序列信息不清楚导致了非特异性扩增； 扩增的基因为多基因家族的成员；mRNA不同的拼接方式造成的。
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