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EA0104 RT-EMA Exo Kit

Cat. No. EB0104
Source /
Appearance Liquid
Endotoxin /
Purity /
Consult Immediately
Detail

RT-EMA   Exo Kit

Cat#EA0104

Description

This kit provides the reagents required for isothermal amplification of nucleic acids with high sensitivity and strong specificity.Pre-operation time is short, reaction temperature (37-42°C) is easily met, and exponential amplification can be completed within 20 minutes.Suitable for RNA templates with a minimum lower limit of detection of 10-100 copies/reaction.

Product Composition

Name

Size

RPA reaction buffer

100 T

Enzyme Mix

100 T

PEG

100 T

Trehalose

100 T

Positive control template

10 μL

Positive control primers + probes

50 μL

Activator

100 T

Storage Conditions

Store at -80. Avoid exposure to frequent temperature changes.

Stability

When placed at -80 for 3 months, the enzyme application activity did not decrease significantly (data update).

Freezing and thawing for 5 times showed no significant decrease in enzyme activity.

The enzyme application activity did not decrease significantly after being placed at 4 for 7 days (data update).

After lyophilization, the enzyme application activity did not decrease significantly.

Quality Control

Endonuclease Activity

The 50 μL reaction system consisted of 10 μL Reaction system mix and 500 ng of single-stranded RNA were incubated at 37℃ for 4 hours. Nucleic acid endonuclease activity was detected using agarose gel electrophoresis.

RNA hydrolase activity

The 50 μL reaction system contained of 10 μL Reaction system mix and 500 ng of single-stranded RNA were incubated at 37℃ for 4 hours.The RNA hydrolase activity was detected using agarose gel electrophoresis.

Host Residual DNA

Escherichia coli 16S rDNA was used as the target to detect the residual amount of host DNA in Reaction system mix by fluorescence quantitative PCR.

Experimental Procedures

1. Add RPA reaction buffer, enzyme mix, PEG, Trehalose and primer probe according to the following system.

Component

volumes

RPA reaction buffer

8 μL

dNTP (25 mM)

0.4-1.5 μL

Enzyme Mix

10 μL

PEG

14 μL

Trehalose

5μL

Forward and reverse primers (10 μM) + probe (5 μM)

2+2+1 μL

Template

4 μL

Activator

2 μL

H2O

UP to 50 μL

2. Close the lid and mix with a vortex oscillator at low speed for 30 seconds, then centrifuge in a microcentrifuge for 10 seconds and repeat the mixing-centrifugation 3 times;

3. Open the lid to add template, add 2 μL activator to the lid of each reaction tube, close the lid and centrifuge, then mix-centrifuge 1 time;

4. Incubate in a holding apparatus at 42°C for 30 minutes.

Examples of applications

RPA-1.jpgRPA-2.jpg

Figure 1. RT-RPA performance evaluation.(A) Comparison of RT-RPA with competitor, Template: RNA virus, Template amount: 105, 104, 103 and 102 copies/reaction. (B) RT-RPA performance comparison before and after lyophilisation, Template: RNA virus, Template amount: 104 and 103 copies/reaction.

Others

Principles of designing target amplification regions and primers

The target amplification should be longer than 70bp but not more than 500bp, preferably around 150-250bp, the range that works best;

The distribution of bases in the target amplification region should be relatively homogeneous, with GC content between 40% and 60%, with low frequency of neighbouring and close positions containing the same bases, and as far as possible free of forward/reverse repeats and palindromic sequences.

Primer lengths are generally around 30-35 nucleotides, with the longest generally not exceeding 42 nucleotides;

The first 3-5 nucleotides at the 5' end of the primer should avoid consecutive base G, while base C/T favours nucleic acid amplification;

The presence of base G/C in the last 3 nucleotides at the 3' end of the primer favours amplification;

The total GC content of the primers should be between 30% and 70%, and the Tm value between 50-100.

Screening of primers

The design of isothermal amplification primers has a large impact on the amplification efficiency, if the primers are required to be high, they can be screened in multiple steps, and the primer design can be carried out according to the figure on the right.

Basic primer screening process

Select the best reverse primer by pairing either forward primer with a candidate reverse primer;

Select the best forward primer by pairing the best reverse primer with the candidate forward primer;

Screening and verification conditions

Reaction conditions for screening without using optimal parameters can reduce template copy number and shorten reaction time;

Primer concentration can be optimised between 150nM-600nM;

Primer screening can be done directly using agarose gel to observe whether the target fragment can be amplified.