Second Strand cDNA Synthesis Kit
The starting material of the cDNA second strand synthesis kit is the RNA DNA hybrid strand that has completed the first strand cDNA synthesis.
The RNA in the RNA DNA heterozygous chain was cleaved using RNase H, and then DNA Polymerase I was used to replace the RNA chain through a nick translation reaction to synthesize the second strand of cDNA. The schematic diagram is as follows:
CDNA Second Strand Synthesis Kit
Ready to use, containing all reagents for synthesizing the second strand of cDNA, including RNase H, E.coli DNA polymerase I, and DNA ligase.
One tube operation eliminates the need for purification each time and does not lose valuable samples.
The obtained double stranded cDNA can be directly used for subsequent routine PCR, real-time PCR, cDNA library construction, suppression subtractive hybridization, etc.
This reagent kit is sufficient for 10 times 80 μ The second strand synthesis reaction of cDNA in the L system.
Component specifications
five × CDNA Second Strand Synthesis Buffer 160 μ L
twenty × CDNA Second Strand Synthase Mixture 40 μ L
0.2M EDTA solution 60 μ L
Ultra pure water 1mL
Storage: -20 ℃, valid for 1 year.
1、 Synthesize the first strand of cDNA
This reagent kit does not provide cDNA first strand synthesis reagents. Users need to provide relevant reagents to synthesize cDNA first strands.
2、 Synthetic cDNA second strand
Up to 10 on the ice bath μ The reaction system (reverse transcription system) in which L has completed the synthesis of the first strand of cDNA, and the reagents in the following table are added in sequence, if more than 10 μ L. Please only use 10 μ L.
Ingredient dosage
CDNA first strand synthetic product 10 μ L
Ultra Pure Water 48 μ L
CDNA Second Strand Synthesis Buffer 16 μ L
CDNA Second Strand Synthase Mixture 4 μ L
80 in total μ L. Gently blow and mix well, centrifuge briefly, and keep at 16 ℃ for 2 hours
Self prepared T4 DNA polymerase (see note) 1 μ L
After gently blowing and mixing, continue to heat at 16 ℃ for 30 minutes
0.2M EDTA solution 2 μ L
Note: Cloning double stranded cDNA generally requires flat terminalization and requires T4 DNA polymerase treatment steps. Subsequent treatments such as PCR and SSH generally do not require the flat terminalization of double stranded cDNA, and can skip the T4 DNA polymerase treatment step and directly terminate the reaction with EDTA.
Termination reaction: If subsequent experiments (such as electrophoresis) do not worry about the impact of EDTA, 4 can be directly added μ Terminate the reaction with L 0.2M EDTA solution, and then store the sample at -20 ℃ for a long time. If subsequent experiments (such as PCR amplification) do not worry about the single or double stranded state of cDNA, the reaction can be terminated by holding at 95 ℃ for 10 minutes. If the subsequent experiment is influenced by EDTA and cannot be single stranded DNA, PCR column purification method can be used for purification.
Electrophoresis 5 μ L detects the quality of cDNA. If the cDNA appears blurry within the range of 0.5 to 10Kb, then the cDNA is qualified. as
