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For general laboratory use.
Unit Definition: One unit is defined as the amount of enzyme that incorporates 25 nmol of dNTP into acid insoluble material in 30 minutes at 65°C.
Shipping: shipped on gel packs
Storage Conditions: store at -20 °C
Shelf Life: 12 months
Form: liquid
Concentration: 8 units/μl
Description:
Warm Start Bst Polymerase is derived from Bacillus stearothermophilus DNA Polymerase I, which has 5’→3’ DNA polymerase activity and strong chain replacement activity, but no 5’→3’ exonuclease activity. Warm Start Bst Polymerase is ideally suitable for strand-displacement, Loop-mediated isothermal amplification (LAMP) and rapid sequencing. The polymerase activity is blocked at ambient temperature and switches on automatically at increasing temperature. It inhibits polymerase activity at room temperature to improve reaction efficiency and prevents nonspecific amplification. It allows preparation of the reaction setup at room temperature.Lyophilisation
Warm Start Bst Polymerase – glycerol free is recommended for preparation of lyophilised LAMP or RT-LAMP mixes.
Glycerol is replaced by a cryoprotectant which does not affect the lyophilisation of the reagent mixture.Content:
Component Cap PCR-542S PCR-542L Warm Start Bst Polymerase red 250 μl 5 x 250 μl Warm Start Bst Buffer yellow 625 μl 5 x 625 μl MgSO4 Solution green 375 μl 5 x 375 μl Detection
Although some methods have been developed to visualize DNA amplification by basic equipment or even the naked eye (increase of turbidity, color change of added dyes, hybridisation to gold-bound ss-DNA) in general, real-time detection of the DNA amplification by a fluorescent DNA-intercalator dye is recommended. Addition of a fluorescent DNA Stain to the assay allows a sensitive measurement of the increasing amount of DNA without influencing the reaction. The setup can also be combined with ROX reference dye (PCR-351) to allow a signal normalisation in real-time PCR instruments that are compatible with the evaluation of the ROX signal.Assay design
Isothermal amplification is an extremely sensitive detection method and care should be taken to avoid contamination of set-up areas and equipment with DNA of previous reactions. A common problem is amplification in no-template controls due to1. carry-over contamination or
2. amplification of unspecifically annealed
primers or primer dimer formations.As sensitivity and non-template amplification of in-silico designed primers may vary, the evaluation of 2-4 real primer sets before choosing a final set is recommended.
Assay set-up
Depending on the detection method and machine a reaction volume of 20-50 μl is recommended for most applications. Pipet with sterile filter tips and perform the set-up in an area separate from DNA preparation or analysis. No-template controls should be included in all amplifications.
First, prepare a 10x conc. primer pre-mix as follows:primer stock conc. final conc. 100 μl 1 ml FIP 100 μM 16 μM 16 μl 160 μl BIP 100 μM 16 μM 16 μl 160 μl F3 100 μM 2 μM 2 μl 20 μl B3 100 μM 2 μM 2 μl 20 μl Loop F 100 μM 4 μM 4 μl 40 μl Loop B 100 μM 4 μM 4 μl 40 μl PCR-grade Water 56 μl 560 μl Then set-up the isothermal amplification assay:
component stock conc. final conc. 20 μl 50 μl Warm Start Bst Buffer 10x 1x 2 μl 5 μl MgSO4 Solution 100 mM 6 mM 1.2 μl 3 μl dNTP Mix
(NU-1006)10 mM each 1.4 mM 2.8 μl 7 μl Green-
Fluorescent
DNA Stain
(PCR-378)100 μM 0.8 mM 0.16 μl 0.4 μl Primer Mix 10x 1x 2 μl 5 μl Warm Start Bst Polymerase 8 units/μl 0.32 units/μl 0.8 μl 2 μl Template
DNA<500 ng/assay x μl x μl PCR-grade
Water
(PCR-258)fill up to 20 μl fill up to 50 μl Amplification and detection
- Use a specific detection instrument for isothermal amplification or a real-time PCR cycler to run the assays
- Set the instrument to a constant incubation temperature between 60 to 65°C (depending on the primer annealing temperature)
- Measure the fluorescence intensity at an interval of 1 min for up to 30 min.
Trouble shouting
If amplification in no-template controls occurs the following points should be reviewed.Cross contamination from environments
- Clean equipment and areas with “DNA Away” solution
- Replace reagent stocks and pre-mixes with new components
- Stop reactions at an earlier point of time before non-template amplification occurs
Carry-over contamination from previous reaction products
- Avoid opening reaction vessels after amplification
- Use separate preparation area and equipment if post-reaction processing is necessary
Non-template amplification from primers
- Increase incubation temperature stepwise by 1-2 °C
- Design a new set of primers for the target sequence
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产品组分
组分内容
型号 规格 储存温度
热启动无甘油Bst聚合酶(8U/μL)
PCR542-01 2000Units -20°C 热启动无甘油Bst聚合酶(8U/μL)
PCR542-02 10000Units -20°C 操作手册
1 1 室温
*注意：在使用之前，应将溶液离心至管底。
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保存建议 推荐蓝冰或冰袋运输。当您收到产品后，按照说明书建议保存各组分。 警告 本品仅供科研使用，请勿用于临床与诊断。 -
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