Saphir RT-LAMP试剂盒

Saphir RT-LAMP试剂盒，Saphir RT-LAMP Kit，可合成与RNA模板互补的单链DNA分子（cDNA），随后进行环介导的含绿色荧光DNA染色剂的快速等温RNA扩增试剂盒。更多视频请关注视频号【艾维缔】。哔哩哔哩【IVDSHOW】。抖音【军哥聊表观】。

关键词

Saphir RT-LAMP试剂盒

所属分类

分子生物学

目录号

PCR540

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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 cycles
store dark

Shelf Life: 12 months

Form: liquid

Spectroscopic Properties: λ_exc 483 nm, λ_em 503 nm (dye bound to DNA)

Content:

Component	Cap	PCR-540S	PCR-540L
RT-LAMP Buffer	yellow	1 ml	5 x 1 ml
RT-LAMP Enzymes	red	200 μl	5 x 200 μl
RT-LAMP Dye	green	160 μl	5 x 160 μl
PCR-grade Water	white	1.2 ml	3 x 1.2 ml

Detection
Saphir RT-LAMP Kit synthesizes single-stranded DNA molecules (cDNA) complementary to the RNA template followed by loop-mediated isothermal DNA amplification. The kit additionally contains a green-fluorescent DNA stain that intercalates into DNA during the amplification process and allows the direct quantification of target RNA by fluorescence detection (analogous to real-time RT-PCR).

Assay design
Isothermal ampliﬁcation is an extremely sensitive detection method and special care should be taken to avoid contamination of set-up areas and equipment with RNA/DNA of previous reactions.Preventing amplification in no-template controls due to carry-over contamination or ampliﬁcation of unspecifically annealed primers or primer dimer formations can be challenging.

Primer design
Typically, 4 different primers are used to identify and amplify a distinct gene region allowing the specific amplification of a target gene. An additional pair of primers further accelerates the amplification allowing to cut down the total detection time to 10-20 min.
The manual design of primers may be difficult due to the complex reaction sequence. To simplify the design process the use of a primer design software is recommended.
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
A reaction volume of 20-50 μl is recommended for most applications. Pipet with sterile ﬁlter tips and perform the set-up in an area separate from RNA/DNA preparation or analysis. No-template controls should be included in all ampliﬁcations.
First, prepare a 10 x conc. primer pre-mix. Second, set-up the isothermal ampliﬁcation assay:

component	stock conc.	final conc.	20 μl	50 μl
RT-LAMP Buffer	4 x	1 x	5 μl	12,5 μl
RT-LAMP Enzymes	20 x	1 x	1 μl	2,5 μl
RT-LAMP Dye	25 x	1 x	0.8 μl	2 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

Use a specific detection instrument for isothermal amplification or a real-time PCR cycler to run the assays
Set the instrument to a constant incubation temperature between 60 to 65°C (depending on the primer annealing temperature)
Select the FAM channel of the real-time instrument
Measure the fluorescence intensity at an interval of 1 min for up to 30 min.

Trouble shooting
If amplification in no-template controls occurs the following points should be reviewed.

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

产品组分

组分内容	型号	规格	储存温度
Saphir RT-LAMP试剂盒	PCR540-01	200次	-20°C
Saphir RT-LAMP试剂盒	PCR540-02	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。

文件资源

营销中心

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。