NZY First-Strand cDNA Synthesis Flexible Pack (MB40001)

Refrigerated package

199,00

200 reactions

199,00

Description: NZY First-Strand cDNA Synthesis Flexible Pack provides robust transcription of RNA as it includes all the necessary components to synthesize first-strand cDNA from an RNA template. This optimized pack includes the following components: our modified recombinant form of the Moloney Murine Leukemia Virus (M-MuLV) Reverse Transcriptase (RNase H minus) that synthesizes the complementary DNA strand using either RNA or single-stranded DNA as template in a wide range of temperatures (50-85°C); an optimized Reverse Transcriptases buffer; a NZY Ribonuclease Inhibitor for RNA protection against degradation due to ribonuclease contamination; a dNTPs mix; random hexamers and oligo(dT)18 primers; and finally, an E. coli RNase H to specifically degrade the RNA template in cDNA-RNA hybrids after the first-strand cDNA synthesis. This flexible pack offers the possibility to choose the primer to initiate the reaction: you can choose either the primers provided in separate tubes, or a gene-specific primer. The pack is sufficient for up to 200 first-strand cDNA synthesis reactions and it is optimized for RT reactions over a wide range of total RNA concentrations from 10 pg to 5 μg. Downstream applications include real-time PCR, standard PCR and microarrays.

Features:
– Provides high yields of full-length cDNA products
– Formulated to increase sensitivity in RT-qPCR
– Primer type: oligo(dT)18 and random hexamers
– Starting material: 10 pg to 5 µg of total RNA
– Thermostable – working temperature range 50-85 ºC
– Convenient and reliable

Applications:
– First-strand cDNA synthesis for use in real-time quantitative RT-PCR (RT-qPCR)
– Two-step RT-PCR assays
– Ideal for high GC-content templates due to the optimum activity at 50 ºC

Specifications:
– Storage conditions: Store all kit components at -20 ºC
– Shipping conditions: Shipped with dry ice

Components:
– NZY Reverse Transcriptase (200 U/μL)
– 10x Reaction buffer for Reverse Transcriptases
– NZY Ribonuclease Inhibitor (40 U/µL)
– dNTP Mix (10 mM each)
– Random hexamer mix (50 ng/µL)
– Oligo(dT)18 primer mix (50 μM)
– NZY RNase H (E. coli) (5 U/μL)
– DEPC-treated H2O

MSDS EN
MSDS PT
Product Brochure EN
Certificate of Analysis YL091
Certificate of Analysis YL092
Certificate of Analysis YL093
Certificate of Analysis YL101
Certificate of Analysis YL102

1. Which components of the NZY First-Strand cDNA Synthesis Flexible Pack for RT-PCR are sold separately? 
The following components of the kit are available for purchase as individual products:
NZYReverse Transcriptase (Cat. No. MB124)
NZYRibonuclease Inhibitor (Cat. No. MB084)
Oligo (dT)18 primer mix (Cat. No. MB12801)
Random hexamer mix (Cat. No. MB12901)
NZYRNase H (E. coli) (Cat. No. MB085)

2. Should I treat the synthesized cDNA with RNase H before PCR?
Addition of RNase H after first-strand synthesis will degrade the RNA used as template, by removing it from the cDNA:RNA hybrid molecule. The RNA is still present when using RNase H versions of reverse transcriptase, as is the case of the NZYReverse Transcriptase. Presence of RNA during PCR could inhibit annealing of the primers to the cDNA and then affect the amplification reaction, especially for long fragments. However, the 95°C denaturing step could cause RNA degradation of the RNA-cDNA hybrids and therefore RNase H treatment may not be necessary. We recommend performing RNase H digestion before PCR when using lower levels of template or when amplifying long fragments.

 3. How much synthesized cDNA should be used in a PCR reaction?
Do not exceed 10% of the final PCR reaction volume. The volume of cDNA used will depend on the amount of RNA used as template for first-strand synthesis, as well as the abundance of the target gene.

 4. Little or no RT-PCR/RT-qPCR amplification product is observed. What should I do?
This may result from several factors, such as:
a) RNA damage or degradation. Analyze RNA by denaturing gel electrophoresis to verify nucleic acid integrity. Use aseptic conditions while working with RNA to prevent RNase contamination. Replace RNA if necessary.
b) Presence of RT inhibitors. Some inhibitors of RT enzymes include: SDS, EDTA, glycerol, sodium phosphate, spermidine, formamide and guanidine salts. They can be problematic if present in smaller reaction volumes. If necessary, remove inhibitors by ethanol precipitation of the RNA preparation before use; wash the pellet with 70% (v/v) ethanol.
c) Not enough starting RNA. Increase the concentration of starting RNA.

5. What to do when unexpected bands are observed after electrophoretic analysis of PCR products using the synthesized cDNA as template?
This may result from several factors, such as:
a) Contamination by genomic DNA. To test if products were derived from DNA, perform a no-RT control during first-strand cDNA synthesis. If amplification products are detected after the PCR reaction in the absence of reverse transcriptase, it may be necessary to eliminate residual genomic DNA from the RNA sample. A RNAse-free DNase may be used (pre-treatment RNA). The DNase volume should not exceed 10% of the total reaction volume.
b) Non-specific annealing of primers. Adjust annealing conditions and/or use a DNA polymerase with hot-start capacity (e.g. Supreme NZYTaq II DNA polymerase, cat. No. MB355). Optimize primers and magnesium concentration.
c) Primer-dimers. Adjust annealing conditions and/or design another set of primers without complementary sequences. Include a positive control in the PCR, i.e. a template that will be correctly amplified with the primers that you designed.

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