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                    Products > Virus-related Products > Gold Reverse Transcriptase R3001 R3002

                    Gold Reverse Transcriptase R3001 R3002

                    Gold Reverse Transcriptase

                    For research use only

                    Components  

                    Component

                    R3001 (2,000 U)

                    R3002 (10,000 U)

                    5 × Gold Buffer

                    500 μl 

                    500 μl 

                    Gold Reverse Transcriptase (200 U/μl)

                    10 μl  

                    50 μl  

                     

                    Storage

                    This reagent should be kept at -30~15°C.

                     

                    Description

                    Gold Reverse Transcriptase is a new reverse transcriptase obtained by in vitro molecular evolution based on M-MLV (RNase H-) Reverse Transcriptase. Gold Reverse Transcriptase has further significantly improved thermal stability, with a half-life of more than 4 hours at 50°C and stable reaction time at 55°C, which is very suitable for reverse transcription of RNA templates with complex secondary structure. In addition, Gold Reverse Transcriptase adds multiple point mutations, further enhancing template affinity and progressiveness, resulting in a significant increase in full-length cDNA synthesis, resulting in up to 20 kb of cDNA. It has higher tolerance to common reverse transcriptional inhibitors and is very suitable for reverse transcriptional reaction of plant tissue RNA rich in polysaccharide polyphenols.

                     

                    Unit Definition

                    Poly (rA)·Oligo (dT) was used as the template/primer. At 37°C for 10 min, the amount of enzyme required to add 1 nmol dTTP as an acid-insoluble substance was defined as 1 unit of activity (U).

                     

                    Quality Control

                    Exonuclease residue detection: 200 U of this product and 50 pmol single-stranded DNA substrate were incubated at 37°C for 16 h, and the DNA electrophoresis band did not change after denaturation PAGE electrophoresis.

                    Detection of endonuclease residue: 200 U of this product and 0.3 μg of pBR322 DNA were incubated at 37°C for 4 h, and the electrophoresis bands of the plasmids were not changed by agarose gel electrophoresis.

                    RNase residue detection: the product of 200 U and 1 μg of 293 cell RNA were incubated at 37°C for 30 min, and the electrophoresis band of RNA was unchanged by agarose gel electrophoresis.

                    E.coli DNA residue detection: the nucleic acid residue in 60 U was detected by E. coli gDNA-specific TaqMan qPCR, and the residue of E. coli genome was less than 10 copies.

                    Functional detection 1: 200 U enzyme was added to the reverse transcription system, 1 μg HeLa cells total RNA was used as template, Oligo (dT)23 as primer, and the reaction was conducted at 50°C for 45 min.1/10 cDNA product was taken for PCR amplification of VIN gene. Agarose gel electrophoresis with EB staining showed a single 4.6 kb band.

                    Functional detection 2: 200 U enzyme was added to the reverse transcription system, 1 pg HeLa cell total RNA was used as template, Oligo (dT)23 as primer, and the reaction was conducted at 50°C for 30 min.1/10 cDNA product was taken for PCR amplification of GAPDH gene. Agarose gel electrophoresis with EB staining showed a single 550 bp band.

                    Functional detection 3: 500 ng HeLa cells total RNA as template, Oligo (dT)23VN as primer, 50°C reaction for 45 min.1/10 cDNA product was taken for PCR amplification of the Polε gene.Agarose gel electrophoresis, EB staining, a single 7.1 kb (gc-rich) band can be seen.

                     

                    Matters Needing Attention

                    Prevent RNase contamination

                    Please keep the experimental area clean.Clean gloves and masks should be worn during operation.RNase-free shall be ensured for the centrifugal tubes, pipetting tips and other consumables used in the experiment.

                    Primers selection

                    The follow-up experiment is PCR

                    If the template is of eukaryotic origin, Oligo dT is generally preferred, paired with the 3 'Poly A tail of eukaryotic mRNA for maximum yield of full-length cDNA.

                    Gene specific primers (GSP) have the highest specificity.However, in some cases, the GSP used for PCR reaction cannot effectively guide the synthesis of the first strand of cDNA.At this point, the reverse transcription can be redone using Oligo dT or Random hexamers.

                    Random hexamers have the lowest specificity, and all RNA, including mRNA, rRNA, and tRNA, could be used as templates for Random hexamers.Random hexamers can be used as a primer when the target region has a complex secondary structure or a high GC content, or when the template is prokaryotic and the use of Oligo dT or gene-specific primers (GSP) cannot effectively guide cDNA synthesis.

                    The follow-up experiment is qPCR

                    Oligo dT mixed with Random hexamers resulted in the same cDNA synthesis efficiency in each region of the mRNA, helping to improve the authenticity and repeatability of the quantitative results.

                     

                    Protocol

                    The follow-up experiment is PCR

                    1. Denaturation of RNA template

                    Prepare the following mixture in RNase-free centrifugal tube:

                    Component

                    Amount

                    Oligo (dT)23VN (50 μM) 

                    or Random hexamers (50 ng/μl)

                    or Gene Specific Primers (2 μM)

                    1 μl

                    Total RNA

                    or Poly A+ RNA

                    10 pg - 5 μg

                    10 pg - 500 ng

                    RNase-free ddH2O

                    to 13 μl

                    After being heated at 65°C for 5 min, quickly place the mixture in an ice bath for quenching, and place on ice for 2 min.

                    RNA template denaturation helps to open the secondary structure, which can greatly increase the production of the first strand of cDNA.For cDNA fragments longer than 3 kb, do not omit the denaturation step.

                    2. Prepared the first cDNA synthesis reaction solution

                    Component

                    Amount

                    The mixture from the previous step

                    13 μl

                    5 × Gold Buffer

                    4 μl

                    dNTP Mix (10 mM each)

                    1 μl

                    Gold Reverse Transcriptase (200 U/μl)

                    1 μl

                    RNase inhibitor (40 U/μl)

                    1 μl

                    Blow gently with a pipette and mix well.

                    2. Conduct the first cDNA synthesis reaction according to the following conditions

                    Temperature

                    Time

                    25°Ca

                    5 min

                    50°Cb

                    45 min

                    85°C

                    2 min

                    a. This step is required only when using Random hexamers;This step is omitted when using Oligo (dT)23VN or Gene Specific Primer.

                    b. If the template has a complex secondary structure or a high GC region, the reaction temperature can be raised to 55°C, which can help increase the yield.

                    The product can be used immediately for PCR reaction or stored at -20°C and used within half a year.For long-term storage, it is recommended to store separately at -70°C.Repeated freeze-thaw of cDNA should be avoided.

                     

                    The follow-up experiment is qPCR

                    1. Prepared the first cDNA synthesis reaction solution

                    Prepare the following mixture in RNase-free centrifugal tube:

                    Component

                    Amount

                    5 × Gold Buffer

                    4 μl

                    dNTP Mix (10 mM each)

                    1 μl

                    Gold Reverse Transcriptase (200 U/μl)

                    1 μl

                    RNase inhibitor (40 U/μl)

                    1 μl

                    Oligo (dT)23VN (50 μM) 

                    1 μl

                    Random hexamers (50 ng/μl)

                    1 μl

                    Total RNA

                    or Poly A+ RNA

                    10 pg - 1 μg

                    10 pg - 100 ng

                    RNase-free ddH2O

                    to 20 μl

                    Blow gently with a pipette and mix well.

                    2. Conduct the first cDNA synthesis reaction according to the following conditions

                    Temperature

                    Time

                    25°C

                    5 min

                    50°C*

                    15 min

                    85°C

                    2 min

                    * If the template has a complex secondary structure or a high GC region, the reaction temperature can be raised to 55°C, which can help increase the yield.

                    The product can be used immediately for PCR reaction or stored at -20°C and used within half a year.For long-term storage, it is recommended to store separately at -70°C.Repeated freeze-thaw of cDNA should be avoided.


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