Isothermal Mastermix Selection Guide
- OptiGene’s Isothermal Mastermixes allow real-time fluorescence detection of amplified DNA and RNA on the Genie® II and Genie® III platforms.
- Mixes are also suitable for generic qPCR instrumentation.
- An anneal curve can be generated post amplification to confirm the amplification product; eliminating the need for gel electrophoresis or turbidity detection; essential for a closed-tube system.
Isothermal mastermixes are suitable for end-point detection but this practice is strongly discouraged due to the extremely high probability of contamination when opening the reaction tube.
- OptiGene’s isothermal mastermixes are fully licensed for LAMP by Eiken Chemical Company.
- OptiGene’s isothermal mastermixes are suitable for alternative isothermal amplification technologies.
- Proprietary and patented reagents offer the fastest reaction times available.
- OptiGene’s DNA polymerases are also available separately.
ISO-001 Isothermal Master Mix - 400 reactions
ISO-001nd Isothermal Master Mix, no dye - 400 reactions
ISO-002 Isothermal Master Mix - 2000 reactions - 5 x 400rxn kits
ISO-002nd Isothermal Master Mix, no dye - 2000 reactions - 5 x 400rxn
GspM and GspM2.0 LF DNA polymerases
GspM LF DNA polymerase, isolated from Geobacillus sp. M, is functionally equivalent to Bst LF DNA polymerase and can be directly substituted for that enzyme.
GspM2.0 is an engineered variant of GspM that has greatly increased speed. It too can be directly substituted for Bst.
A comparison between GspM, GspM2.0, Bst and Bst2.0 in a fluorescent detection LAMP reaction run on the Genie® II instrument is shown in the figure to the left.
|GSPM-002||8,000u GspM DNA polymerase @ 8u/µl|
|GSPM-002HC||8,000u GspM DNA polymerase @ 100u/µl|
|GSPM-003||40,000u GspMDNA polymerase @ 8u/µl|
|GSPM-003HC||40,000u GspM DNA polymerase @ 100u/µl|
|GspM2-002||8,000u GspM2.0 DNA polymerase @ 8u/µl|
|GspM2-002HC||8,000u GspM2.0 DNA polymerase @ 100u/µl|
|GspM2-003||40,000u GspM2.0 DNA polymerase @ 8u/µl|
|GspM2-003HC||40,000u GspM2.0 DNA polymerase @ 100u/µl|
Reverse Transcription LAMP
GspSSD LF DNA polymerase, as used in the ISO-001 master mix, has good reverse transcriptase (RT) activity. GspM2.0 DNA polymerase has poor RT activity whilst the unmodified GspM DNA polymerase has no RT activity.
The addition of 0.125u of AMV reverse transcriptase will enhance the RT activity further, decreasing the reaction time, but the overall assay sensitivity is identical with or without AMV.
A comparison between ISO-001, GspM, GspM2.0, Bst and Bst2.0 in a fluorescent detection human B-actin RT LAMP reaction, run on the Genie® II instrument, is shown in the figure to the left.
GSPSSD-002 8,000u GspSSD DNA polymerase @ 8u/µl
GSPSSD-002HC 8,000u GspSSD DNA polymerase @ 100u/µl
GSPSSD-003 40000u GspSSD DNA polymerase @ 8u/µl
GSPSSD-003HC 40,000u GspSSD DNA polymerase @ 100u/µl
Thermostable LF DNA polymerase
Tin exo-LF DNA polymerase, isolated from Thermodesulfatator indicus, has strong strand displacement activity and is thermostable for 10 minutes at 93ºC.
The full length unmodified enzyme possesses both 5’-3’ exonuclease and 3’-5’ exonuclease activities. Tin exo-LF DNA polymerase has been engineered to remove the 5’-3’ exonuclease domain and inactivate the 3’-5’ exonuclease activity. The enzyme has no reverse transcriptase activity.
The thermostability of Tin LF DNA polymerase can be seen in the figure on the left. Tin LF DNA pol can be heated to 93oC for 10 minutes with no loss of activity observed. Template denaturation using a heat step prior to 65C isothermal amplification has been shown to increase target specificity and reduce overall amplification times.
TIN-001 5,000u Thermodesulfatator indicus exo- LF DNA polymerase @
TIN-002 25,000u Thermodesulfatator indicus exo- LF DNA polymerase @
TIN-002HC 25,000u Thermodesulfatator indicus exo- LF DNA
polymerase @ 100u/µl
|ISO-001||Isothermal Master Mix - 400 reactions of 25µl — includes intercalating dye|
|ISO-001nd||Isothermal Master Mix - 400 reactions of 25µl — no intercalating dye|
|ISO-002||Isothermal Master Mix - 2000 reactions of 25µl — includes intercalating dye|
|GSPM-001||1600u GspM2.0 DNA polymerase @ 8u/µl|
|GSPM-002||8000u GspM LF DNA polymerase @ 8u/µl|
|GSPM2-002||8000u GspM2.0 LF DNA polymerase @ 8u/µl|
|GSPM2-002HC||8000u GspM2.0 LF DNA polymerase @ 100u/µl|
|TIN-001||5,000u Thermodesulfatator indicus exo- LF DNA polymerase @ 8u/µl|