SPEED cDNA Synthesis Kit

SPEED cDNA Synthesis Kit

SKU Description Size Price Shipping  
LYM1010 SPEED cDNA Synthesis Kit 25 rxns $96 2-5 Days Add To Shopping Cart
LYM1011 SPEED cDNA Synthesis Kit Plus with Higher Efficiency 25 rxns $222 Immediate Add To Shopping Cart
Datasheet:
The Kit is for synthesis of first strand cDNA from RNA templates. The kit utilizes a mutated recombinant M-MuLV Reverse Transcriptase which exhibits minimal RNaseH activity. Due to this feature, full-length cDNA can be synthesized from RNA templates that are up to 9 kb long. Recombinant RNasin™ Ribonuclease Inhibitor supplied with the kit effectively can protects RNA template from degradation.The kit is supplied with both oligo(dT)15 and random hexamer primers. Gene-specific primers may also be used with the kit.

Size   

25 Reactions

Usage   

First strand cDNA synthesis for RT-PCR.
Construction of cDNA libraries.
Generation of probes for hybridization.

Components

RTase (200U/µl)                      25µl
Oligo(dT) (10µM)                     40µl
Random Primers (10µM)         40µl
dNTP (10mM)                          40µl
RNasin (40U/ml)                      15µl
5X RT buffer                           150µl
RNase-free Water                    1ml
 

Storage   

Keep at -20°C

Protocol

1. Thaw template RNA and all reagents on ice. Mix each solution by vortexing, and centrifuge briefly to collect residual liquid from the sides of the tubes.

2. Prepare the following reaction mixture in a PCR tube on ice:

                        Total RNA                                                     0.5-5µg
                             or Poly(A)+RNA                                     50ng-0.5µg
                        Oligo(dT) or Random Primer (10uM)            1µl
                        dNTP (10mM)                                               1µl
                        5X RT Buffer                                                 4µl
                        RNasin (40U/ul)                                         0.5µl
                         RTase (200U/ul)                                           1µl
                        Water                                                            20µl


3. Incubate at 25°C for 10 minutes if random primer is used. Omit this step if Oligo(dT) primer or sequence-specific primer is used.

4. Incubate the mixture at 42°C for 60 minutes.

5. Stop the reaction by heating at 85°C for 5 minutes. The newly synthesized first-strand cDNA can be used directly for amplification by PCR.
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