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DNA/RNA Modifying Enzymes

Maxima® Reverse Transcriptase

- Thermal inactivation at 85°C in 5 min
- Recombinant enzyme
- LO certified
- Store at -20°C

Catalog#	Size, concentration	Supplied with:	Certificate of Analysis	MSDS
Catalog#	Size, concentration	5X RT Buffer	Certificate of Analysis	MSDS
EP0741	2000 u (200 u/µl)	1.00 ml	EP0741
EP0742	10,000 u (200 u/µl)	1.00 ml	EP0742
EP0743	4 x 10,000 u (200 u/µl)	2 x 1.00 ml	EP0743

Product information

Features

High yields of full-length cDNA up to 20 kb.
Active up to 65°C.
Thermostable – 90% active after incubation at 50°C for 60 minutes.
Efficient – complete cDNA synthesis in 15-30 minutes.
High sensitivity – reproducible cDNA synthesis from a wide range of total RNA quantities (1 pg – 5 μg).
Incorporates modified nucleotides.

Applications

Two step RT-PCR.
Two step RT-qPCR.
First strand cDNA synthesis.
Construction of full length cDNA libraries.
DNA labeling.
Primer extension.

Description

Maxima® Reverse Transcriptase (RT) was developed by Fermentas through in vitro evolution of M-MuLV RT. The enzyme possesses an RNA-dependent and DNA-dependent polymerase activity as well as RNase H activity. The engineered enzyme features dramatically improved thermostability, robustness and increased synthesis rate compared to wild type M-MuLV RT.

Maxima® Reverse Transcriptase is capable of reproducible cDNA synthesis from a wide range of input total RNA amounts (from 1 pg to 5 μg) at elevated temperatures (50-65°C), which makes this enzyme an ideal tool for two step RT-qPCR (see Fig.5).

Due to its high thermostability (see Fig.1), the enzyme maintains full activity during the entire reverse transcription reaction, generates the highest yields of cDNA (see Fig.3) and is able to synthesize even very long RNA transcripts up to 20 kb (see Fig.2). The reaction temperature can be increased up to 65°C for efficient transcription of RNA regions with a high secondary structure or to improve specificity using gene specific primers. Due to its increased synthesis rate, the reverse transcriptase reaction can be completed in 15-30 min (see Fig.4).

Source

E.coli cells carrying an engineered pol gene fragment of Moloney Murine Leukemia Virus.

Definition of Activity Unit

One unit of the enzyme incorporates 1 nmol of dTMP into a polynucleotide fraction (adsorbed on DE-81) in 10 min at 37°C.

Enzyme activity is assayed in the following mixture: 50 mM Tris-HCl (pH 8.3), 4 mM MgCl₂, 10 mM DTT, 50 mM KCl, 0.5 mM dTTP, 0.4 MBq/ml [³H]-dTTP, 0.4 mM polyA•oligo(dT)_12-18.

Storage Buffer

The enzyme is supplied in:
50 mM Tris-HCl (pH 7.5), 0.1 M NaCl, 1 mM EDTA, 5 mM DTT, 0.1% (v/v) Triton X-100 and 50% (v/v) glycerol.

5X RT Buffer

250 mM Tris-HCl (pH 8.3 at 25°C), 375 mM KCl, 15 mM MgCl₂, 50 mM DTT.

Quality Control

The absence of endodeoxyribonucleases, exodeoxyribonucleases, phosphatases and ribonucleases confirmed by appropriate quality tests. Functionally tested in first strand cDNA synthesis.

Inhibition and Inactivation

Inhibitors: metal chelators, inorganic phosphate, pyrophosphate and polyamines.
Inactivated by heating at 85°C for 5 min.

Figure 1. High thermostability of Maxima® Reverse Transcriptase at 50°C.
Reverse transcriptases were incubated in 1X reaction buffer. At the indicated time points (5-240 minutes), enzyme activity was determined in a standard activity assay.

Figure 2. Amplification of targets up to 20 kb in two step RT-PCR.
1 µg of total RNA from Jurkat cells (1 and 2) or total RNA from mouse (3 and 4) were used in a reverse transcription reaction with Maxima® Reverse transcriptase and other RTs in the optimal conditions for each enzyme. Synthesized cDNA was used as a template in PCR with the Long PCR Enzyme Mix (#K0181) and primers specific for different genes:
1 – 6.8 kb POLE (human polymerase)
2 – 9.4 kb FBN1 (human fibrillin 1)
3 – 13.3 kb Dmd (mouse dystrophin)
4 – 20.0 kb Neb (mouse nebulin)
M – GeneRuler™ 1 kb Plus DNA Ladder (#SM1331)

Figure 3. High yields of cDNA over a broad temperature range.
Synthesis of ³³P-labeled cDNA was performed using 200 units of each reverse transcriptase, 1 µg of Ambion RNA Millennium™ markers, and oligo(dT)₁₈ primer at various temperatures. Reaction products were analyzed on a 1% alkaline agarose gel.

Figure 4. High cDNA synthesis rate at 50°C.
Synthesis of cDNA was performed at 50°C for 5, 15, 30 and 60 minutes using 1 µg of 7 kb RNA transcript as a template. Reaction products were analyzed on a 1% alkaline agarose gel. Only Maxima® RT was able to complete synthesis of 7 kb transcript in 5 min.

Figure 5. Reproducible RT-qPCR results using Maxima® Reverse Transcriptase.
100 ng-1 pg of total Jurkat cell RNA and a mix of Oligo(dT)₁₈ and random hexamer primers were used with Maxima® RT in 16 replicate reactions. Synthesized cDNA was used as a template in subsequent qPCR with Maxima® SYBR Green/ROX qPCR Master Mix (#K0221) on the ABI 7500 Real-Time PCR instrument. Parallel reactions with Maxima® RT demonstrate reproducible cDNA synthesis and low variability levels (<1% SD/Cq) with a wide range of starting RNA amounts.

Patents, Licenses, Trademarks

Protocols & recommendations

RECOMMENDATIONS FOR USE

ADDITIONAL PROTOCOLS

Guidelines to Avoid RNase Contamination

RNA purity and integrity is essential for synthesis of full-length cDNA, which results in high quality RT-PCR products. Therefore, RNase contamination is always a concern when working with RNA. The RNA quality can be affected by RNase A, which is a highly stable contaminant of any laboratory environment. All components of the kit have been rigorously tested to ensure that they are RNase free. To prevent contamination both the laboratory environment and all prepared solutions must be free of RNases.

DEPC-treat all tubes and pipette tips to be used in the cDNA synthesis or use certified nuclease-free labware.
Use pipettes dedicated for RNA work.
Wear gloves when handling RNA and all reagents, as skin is an common source of RNases. Change gloves frequently.
Use certified reagents, including high quality water (e.g., nuclease-free or DEPC-treated Water).
Use an RNase inhibitor, such as RiboLock™ RNase Inhibitor, to protect template RNA.
Always assess the integrity of RNA prior to cDNA synthesis. For example, if sharp bands of both the human 18S rRNA (runs at approx. 1.9 kb) and the 28S rRNA (runs at approx. 5 kb) are formed during denaturing agarose gel electrophoresis of total RNA, the mRNA in the sample is considered to be intact.

Removal of Genomic DNA from RNA Preparations

Add to an RNase-free tube:

RNA 1 µg

10X reaction buffer with MgCl₂ 1 µl

DNase I, RNase-free 1 µl (1 u)

DEPC-treated Water to 10 µl

Total volume 10 µl
Incubate 30 min at 37°C.
Add 1 µl of 50 mM EDTA and incubate 10 min at 65°C. RNA hydrolyzes if heated in the absence of a chelating agent (1).
Use the prepared RNA as a template for reverse transcriptase.

Note

Do not use more than 1 u of DNase I, RNase-free per µg of RNA.
Reaction mixture can be scaled up for larger amounts of RNA. The recommended final concentration of RNA is 0.1-0.2 µg/µl.
RiboLock™ RNase Inhibitor (#EO0381), typically at 1 u/µl, can also be included in the reaction mixture to inactivate type A RNases potentially present in the initial RNA solution

Reference

Wiame, I., et al., Irreversible heat inactivation of DNase I without RNA degradation, BioTechniques, 29, 252-256, 2000.

First Strand cDNA Synthesis

Master Mix. To prepare several parallel reactions and to minimize the possibility of pipetting errors and contamination, prepare a RT master mix by adding all reaction components except RNA into one vial. Prepare enough master mix for the number of reactions and add one extra to compensate for pipetting errors. Sample template RNA into individual tubes and keep on ice. Aliquote the prepared master mix into tubes with RNA. Mix and briefly centrifuge all components after thawing, keep on ice.

Add into sterile, nuclease-free tube on ice in the order given:

Total volume		14.5 µl
Template RNA	Total RNA or Poly(A) RNA or Specific RNA	1 pg – 5 µg 0.1pg-0.5 µg 0.01 pg – 0.5 µg
Primer	Oligo(dT)₁₈ primer (#SO131) or Random hexamer primer (#SO142) or Gene-specific	0.5 µg (100 pmol) 0.2 µg (100 pmol) 15-20 pmol
dNTP Mix, 10mM each (#R0191)		1 µl (0.5 mM final concentration)
Water, nuclease free (#R0581)		to 14.5 µl

Optional: If RNA template is GC-rich or is known to contain secondary structures, mix gently, centrifuge briefly and incubate at 65°C for 5 min, chill on ice, briefly centrifuge and place on ice.
Add in the indicated order:

5X RT Buffer 4 µl

RiboLock™ RNase Inhibitor(#EO0381) 0.5 µl (20 u)

Maxima® Reverse Transcriptase 1 µl (200 u)

Total volume 20 µl
Mix gently and briefly centrifuge.
If oligo(dT)₁₈ primer or gene-specific primer is used, incubate 30 min at 50°C.
If random hexamer primer is used, incubate 10 min at 25°C followed by 30 min at 50°C. For reverse transcription of GC-rich RNA reaction temperature can be increased up to 60°C.
Terminate the reaction by heating at 85°C for 5 min.

Do not heat inactivate enzyme prior to analysis of long cDNA to avoid cleavage.

The reverse transcription reaction product can be directly used in PCR, qPCR or second strand cDNA synthesis or stored at -20°C for up to one week. For longer storage -70°C is recommended. Avoid freezing/thawing of cDNA.
Use 2 µl of the reaction mix to perform PCR in 50 µl reaction volume.

Recommendations for two-step RT-qPCR

Priming: use a mix of oligo (dT)18 and random primers 25 pmol each per 20 μl reaction.
Incubation: 10 min at 25°C followed by 15 min at 50°C.

Recommendations for long RT-PCR (>5 kb)

Priming: oligo (dT)18 or gene specific primer should be used.
Use 20 u of Maxima® Reverse Transcriptase per reaction. 1X RT buffer can be used to dilute the enzyme just prior to reaction.
Incubation: 30 min at 50°C.

Synthesis of cDNA Probes with High Specific Radioactivity and Non-radioactively Labeled cDNA

I. Synthesis of cDNA probes with high specific radioactivity

Mix and briefly centrifuge all components after thawing, keep on ice.

Add into sterile, nuclease-free tube on ice in the order given:

Total volume		8.5 µl
Template RNA	Total RNA or	1 pg - 5 µg
	Poly(A) RNA or	0.1 pg - 0.5 µg
	Specific RNA	0.01 pg - 0.5 µg
Primers	Oligo(dT)₁₈ (#SO131) or	0.5 µg (100 pmol)
	Random Hexamer (#SO142) or	0.2 µg (100 pmol)
	Gene-specific	15-20 pmol
Water nuclease free (#R0581)		to 8.5 µl

Optional: if RNA template is GC-rich or is known to contain secondary structures, mix gently, centrifuge briefly and incubate at 65°C for 5 min, chill on ice, briefly centrifuge and place on ice.

Add into the same tube in the indicated order:

Total volume	20 µl
5X RT Buffer	4 µl
RiboLock™ RNase Inhibitor (#EO0381)	0.5 µl (20 u)
dGTP, dCTP, dTTP mix, 10 mM each	1 µl
0.1 mM dATP	4 µl
[alpha-³²P]-dATP, 3000 Ci/mmol	1 µl
Maxima® Reverse Transcriptase	1 µl (200 u)

Mix gently and centrifuge to collect all drops.
If Oligo(dT)₁₈ primer or gene-specific primer is used, incubate 30 min at 50°C. If random hexamer primer is used, incubate 10 min at 25°C followed by 30 min at 50°C. For reverse transcription of GC-rich RNA reaction temperature can be increased up to 60°C.
Terminate the reaction by heating at 85°C for 5 min.
Optional. Hydrolyze RNA by the addition of equal volume (25 µl) of 0.6 M NaOH and incubation at 70°C for 30 min.
Remove unincorporated dNTPs by spin column (e.g. GeneJET PCR purification Kit, #K0701) or chromatography on a Sephadex® G-50 column.
Expect specific radioactivity of >10⁷ dpm/µg.

Note

To achieve higher specific activities (over 10⁸ dpm/µg), use up to 100 µCi of [alpha-³²P]-dATP in the labeling mixture. To keep the total reaction volume of 20 µl, vacuum-evaporate 10 µl of [alpha-³²P]-dATP (10 mCi/ml) to 1 µl in a separate tube.

II. Synthesis of non-radioactively labeled cDNA

The protocol above can be used for synthesis of non-radioactive labeled cDNA using biotin-11-dUTP, fluorescein-12-dUTP, DIG-dUTP or aminoallyl-dUTP:

normal dTTP is subsituted with labeled-dUTP at a molar ratio of 1:3 - 1:4,
reaction time is prolonged to 2-6 hours.

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