2x PCR LongNova-RED PCR Master Mix

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1 2x PCR LongNova-RED Components RP85L 100 reactions (50 μl) RP85L reactions (50 μl) 2x PCR LongNova-RED 2 x 1.25 ml 20 x 1.25 ml PCR grade water 2 x 1.5 ml 20 x 1.5 ml Storage & Shiing Storage conditions After thawing, the product will remain stable at +4 C for at least two months. Up to ten freeze/thaw cycles will not compromise product performance. Shiing conditions Shiing on dry or blue ice. For research use only Date of purchase Warranty 12 months from the date of purchase BLIRT S.A. DNA-Gdansk Division

2 RP85L, RP85L-10 2x PCR LongNova-RED

3 RP85L, RP85L-10 2x PCR LongNova-RED 2x PCR LongNova-RED is a 2x concentrated, ready-to-use PCR master mix which facilitates an easy and rapid PCR reaction set up. The 2x PCR LongNova-RED solution contains a reaction buffer, magnesium chloride, dntps and a mixture of thermostable Pwo and Taq polymerases. In order to set up the PCR reaction, all that needs adding to the master mix is the template, the primer set and water. This convenient 2x PCR LongNova-RED master mix reduces the time required to set up PCR reactions and decreases the possibility of contamination, particularly when preparing large numbers of reactions. It is ideal for long range PCR (up to 20 kb) and GC-rich template amplification. The master mix is also sulemented with an inner dye and a density reagent, which permits the direct loading of PCR products to a gel. BLIRT S.A. DNA-Gdansk Division

4 Features and advantages Convenient the LongNova DNA Polymerase in a ready-to-use mix Wide range of product sizes from 2 to 20 kb High proofreading properties (3 5 exonuclease activity) Leaves A overhangs Facilitates high throughput PCR reaction set up Reduced risk of contamination Direct gel loading Alications Very long PCR products High-throughput reactions Molecular cloning Site-directed mutagenesis and other methods requiring high fidelity

5 RP85L, RP85L-10 2x PCR LongNova-RED Protocol 1. Prior to use, thaw the Master Mix solution and other reagents completely, mix thoroughly and spin briefly. 2. Add the following reaction reagents to a sterile nuclease-free PCR Eendorf tube: Reagent Suggested amount per reaction Acceptable final concentrations in reaction mixture 2x PCR LongNova-RED 25 μl 1x 10 µm Forward primer 1 μl µm 10 µm Reverse primer 1 μl µm DNA template ng 10 pg 0.5 µg PCR-grade water fill up to 50 μl fill up to required volume This composition is intended for use as a guide only; conditions will vary from reaction to reaction and may need optimising. 3. Mix the prepared reaction mixture thoroughly by pipetting or vortexing, then spin briefly.

6 4. Place the prepared PCR mixture in a thermal cycler and start the PCR reaction. The table below shows a sample PCR cycling conditions. Step Temperature [ C] Time Initial denaturation 95 C 1 5 min (1) Denaturation 95 C 15 s Annealing C (2) 30 s Extension 68 C 2 20 min (3) Final extension 72 C 5 min Cooling 4 C cycles (4) 1) The initial denaturation time depends on the GC content within the amplified region and the template DNA type. For non-complex templates, such as plasmid DNA or cdna, the initial denaturation step, carried out briefly (1 2 min), is recommended. For more complex templates, such as eukaryotic genomic DNA, a longer initial denaturation step (3 5 min) is required. 2) The annealing temperature depends on the primer sequences and their melting temperature (Tm). The optimal annealing temperature is usually 2 5 C below the Tm of primers. 3) The elongation time depends on the length of an amplified product. Setting 1 min per 1000 bp of the amplified product is recommended. 4) The number of cycles depends on the number of copies of the amplified gene fragment. Thirty cycles is sufficient for low complexity templates. In the case of high complexity templates or less concentrated DNA, the number of cycles should be increased to forty. 5. After reaction has finished, aly the reaction mixtures directly onto a gel.

7 Troubleshooting For problems which may be encountered during PCR reaction set up and analysis, possible causes and solutions see: Notes BLIRT S.A. DNA-Gdansk Division

8 Formulation 2x concentrated PCR reaction buffer, 4 mm MgCl 2 ; 1. 6 m M d N T P s m i x (0.4 mm of each dntp); 0.1 U/µl LongNova DNA Polymerase, red inner dye and density reagent. Quality control Free of unspecific nucleases and DNA contamination. Extensively tested in various long range PCR reactions.

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