Kit Components

Component	EQ003-01	EQ003-02
gDNA Eraser	50 μL	200 μL
10×gDNA Eraser Buffer	50 μL	200 μL
EntiLink™ Reverse Transcriptase	10000U	40000U
5×RT Buffer	0.5 mL	1.0 mL
RNase-Free ddH2O	1.5 mL	1.5 mL
pd(N)9	50 μL	200 μL
oligo dT（18）	50 μL	200 μL
User Manual	1 copy	1 copy

Kit application

1. cDNA library
2. RT-qPCR reaction and RT-PCR
3. Primer
4. RNA sequencing.

Active unit

The product concentration is 200U/μL. A unit of activity (U) is defined as: Poly (A) as the template and Oligo (dT) as the primer, Reaction at 37°C for 10 minutes can mix 1 mole of dTTP into the amount of enzyme required for acid-insoluble substances.

Purity

The purity was greater than 90% by Coomassie blue staining SDS-PAGE. The product was free of endonuclease, exonuclease and RNase contamination.

Self supplied Reagents and items

1. RNase-free 200μL microcentrifuge tube
2. Pipettes and tips (to avoid RNase contamination, RNase-free pipette tips with filter cartridges must be used)
3. Disposable gloves, masks and other protective equipment
4. Constant temperature water bath
5. In RNase-free laboratory operations: Because of the RNase in saliva and skin, wear latex gloves and a mask during the whole process of RNA extraction.

Operation steps

Remove genomic DNA response (on ice):

Reagent	Usage amount
10×gDNA Eraser Buffer	1.0 μL
gDNA Eraser	1.0 μL
Template RNA	0.5-5 μg
RNaser-Free Water	to 10.0 μL

Incubate at 42 ° C for 2 min (or room temperature for 5 min) Store at 4 ° C

Reverse transcription reaction (on ice)

Reagent	Usage amount
Step 1 reaction solution	10.0 μL
oligodT（10 μM） or pd(N)9（10 μM） or Gene Specific Primers （10 μM）*1	1.0 μL
	or 1.0 μL
	or 1.0 μL
RNase-Free ddH2O	to 15.0 μL

After heating the mixture at 70 ° C for 5 min, it was quickly placed on ice for cooling. After a brief centrifugation, add the following components:

Reagent	Usage amount
Step 1 reaction solution
5×RT Buffer	4.0 μL
EntiLink™ Reverse Transcriptase*2	1.0 μL
RNase Inhibitor*3	1.0 μL

The kit provide oligodT and pd(N)9, which can be selected according to experimental needs; Gene Specific Primers need to be prepared by customers

The amount of  Reverse Transcriptase should be reduced to 0.05-0.5 μl when less than 0.5 μg of Total RNA (such as reverse transcription of viral RNA). Otherwise, subsequent PCR amplification may result in non-specific amplification products.

When adding less than 0.5 μg of Total RNA, it is recommended to add 1 μl of RNase Inhibitor

(Cat. No. 010EQ).

Reverse transcription program settings

Temperature	Time
25℃*1	5min
42℃	30min*2
85℃	5min

1 When using pd(N)9, it takes 25℃, 5min. This step can be omitted if oligdT or Gene Specific Primers are used.

*2 To increase the cDNA yield, the reverse transcription time can be extended to 60min.