Multiplex PCR Master (2 conc.)
Detailní popis
For in vitro use only!
Shipping: shipped on blue ice
Storage Conditions: store at -20 °C
avoid freeze/thaw cycles
Shelf Life: 12 months
Form: liquid
Concentration: 2x conc.
Description:
Multiplex PCR Master is specially designed for the set-up of multiplex PCR reactions. It contains an optimized composition of polymerase, nucleotides, MgCl2 and stabilizing components in a specifically developed buffer system allowing the parallel amplification of a multitude of fragments in a single PCR assay.
The master mix contains all reagents (except primer and template) in a 2x concentrated ready-to-use solution.
The kit is recommended for use in routine PCR reactions and highly suitable for multiple target gene amplification in a single tube.
The high specificity and sensitivity of the mix is achieved by a chemically inhibited hot-start polymerase. Its activity is blocked at ambient temperature preventing the extension of nonspecifically annealed primers and primer-dimer formations at low temperatures during PCR setup.
Content:
2x Multiplex PCR Master (red cap)
master mix containing Hot Start Taq polymerase, nucleotides, optimized reaction buffer and stabilizers
PCR grade water (white cap)
Recommended 50 μl PCR assay:
Prepare a master mix of all components except template to reduce pipetting errors. A reaction volume of 20-50 μl per assay is recommended for most PCR cyclers. Pipet with sterile filter tips and perform the setup in an area separate from DNA preparation or analysis. No-template controls should be included in all amplifications.
| component | stock conc. | final conc. | 50 μl assay |
| Multiplex PCR Master | 2x | 1x | 25 μl |
| forward primer 1 | 10 μM | 400 nM | 2 μl |
| reverse primer 1 | 10 μM | 400 nM | 2 μl |
| forward primer 2 | 10 μM | 400 nM | 2 μl |
| reverse primer 2 | 10 μM | 400 nM | 2 μl |
| forward primer ... | 10 μM | 400 nM | 2 μl |
| reverse primer ... | 10 μM | 400 nM | 2 μl |
| Template a) animal genomic DNA b) bacterial genomic DNA c) plasmid and lambda DNA |
- | - | a) 10-200 ng b) 1 - 50 ng c) 1 - 5 ng |
| PCR-grade water | - | - | fill up to 50 μl |
Recommended cycling conditions:
| Initial denaturation |
95 °C | 12 min | 1x |
| Denaturation | 95 °C | 30 sec | 30 - 50x2) |
| Annealing1) | 58 - 64 °C | 40 sec | 30 - 50x2) |
| Elongation3) | 72 °C | 1 min/kb | 30 - 50x2) |
| Final elongation |
72 °C | 5 min | 1x |
1)The optimal annealing temperature (AT) can be calculated for each primer as following:
AT = Tm - 5 °C with Tm = 2 °C x (A+T) + 4 °C x (G+C)
Please note that primers should be designed to show minimal differences in there melting temperatures (Tm).
2)Cycle numbers are recommended as following:
- animal genomic DNA
10 - 50 ng: 35 - 50 cycles
50 - 200 ng: 30 - 45 cycles - bacterial genomic DNA
1 - 5 ng: 35 - 50 cycles
5 - 50 ng: 30 - 40 cycles - plasmid and lambda DNA
1 - 5 ng: 30 - 40 cycles
3)The elongation time depends on the length of the fragments to be amplified. A time of 1 min/kb is recommended.
For optimal specificity and amplification an individual optimization of the recommended parameters may be necessary for each new template DNA and/or primer pair.
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