Back to Products
MINOTECH RT
* Size:


MINOTECH RT

(concentration 200U/μl)

                                technical brochure

 Source: Purified from an E. coli strain carrying a plasmid with M-MuLV reverse transcriptase gene.

Description: High purity reverse transcriptase suitable for first strand  cDNA synthesis. 

Unit definition: One unit is defined as the amount of enzyme required to incorporate 1 nmol of dTTP into acid-insoluble material in a total reaction volume of 50 μl in 10 minutes at 37°C using poly(rA)•oligo(dT)18 as template.

Quality Control Assays:

  • Functional Assay: MINOTECH RT is tested for performance in first strand cDNA synthesis followed by PCR with Taq polymerase. The resulting 1.600 bp PCR product is visualized as a single band on an ethidium bromide-stained agarose gel. MINOTECH RT has been successfully used for synthesis of DNA fragments up to 8.8kb size.
  • Absence of contaminants: Tested extensively for the absence of nicking, endo- and exodeoxyribonucleases and RNases

Guaranteed stability: MINOTECH RT is guaranteed to maintain stability until expiration date.

Recommended First-Strand cDNA Synthesis mixture:

  • 1 µl of oligo(dT)20 (100 µM); or 200–500 ng of oligo(dT) 12-18 ; or
  • 50–250 ng of random primers; or 2 pmol of gene-specific primer
  • 10 ng–2 µg total RNA 
  • 1 µl 10 mM dNTP Mix (10 mM each dATP, dGTP, dCTP and dTTP at neutral pH)
  • Sterile ultrapure water up to 13 µl. 
  1. Heat mixture to 65°C for 5 minutes
  2. Incubate on ice for at least 1 minute
  3. Brief centrifugation and add:
  • 4 µl 5X MINOTECH RT assay buffer
  • 1 µl 0.1 M DTT
  • 1 µl RNase Inhibitor (40 units/µl)
  • 1 µl of MINOTECH RT (~200 units/µl)*

* 400 U of MINOTECH RT can be added to increase yield (for the generation of cDNA >5kb).

  1. Mix gently. If using random primers, incubate tube at 25°C for 5 minutes.
  2. Incubate at 37-42°C for 30–60 minutes.
  3. Heat inactivation step at 70°C for 15 minutes.

Optional (recommended for PCR targets >1kb). Remove RNA complementary to the cDNA, by adding 2 units of E. coli RNase H and incubate at 37°C for 20 minutes.
Recommended PCR mixture: 
10x MINOTECH Taq pol. buf.  5 μl
10mM dNTP mix  1 μl
25μΜ forward primer  1 μl
25μΜ reverse primer  1 μl
cDNA(from first-strand reaction)  2 μl
MINOTECH Taq DNA pol. (5 u/μl)  0.25-0.5 μl
Sterile ultrapure water  Up to 50 μl
 

 

Recommended PCR conditions:
Initial denaturation  94oC, 2min
   

25-35

PCR Cycles 		 Denature  94oC, 45sec
Anneal*  45-68oC, 30sec
Extend  72oC, 1min/kb
Final extension  72oC, 10min
Hold  4oC, indefinitely
 
*Anneal temperature depends on primer Tm