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Cat. No.
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Size
Reactions
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5X
Amplifier
Buffer (dNTP included)
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10X
Primer- mix
|
Amplifier Polymerase
|
Control Genomic DNA
(10 ng/μl)
|
|
280801
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25
|
250 μL
|
50 μL
|
25 μL
|
25 μL
|
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280803
|
100
|
1 mL
|
200 μL
|
100 μL
|
100 μL
|
|
280805
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500
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4x 1.25 mL
|
1 mL
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500 μL
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500 μL
|
Store at -20°C |
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Note: For longer storage, the Amplifier polymerase
should be stored at –70 °C; all other component can be
stored at –20 °C
Reagent for in-vitro laboratory use only |
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| General Description |
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DNase I is purified from bovine pancreas that degrades single-stranded or double-stranded DNA to produce
3-hydroxyl oligonucleotides. This enzyme is used in molecular biology techniques like digestion of DNA, in the
RNA isolation, nick translation and DNase I footprinting. |
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| Storage buffer: |
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The AmpliQ Genomic Amplifier Kit generates an almost unlimited source of DNA for genetic studies. The AmpliQ Genomic Amplifier method exponentially amplifies single or double stranded linear DNA template during an isothermal strand displacement reaction. Amplification of genomic DNA from lysates generates representative, high fidelity DNA (error rate 107) that can be used in various genetic studies. The DNA generated by the AmpliQ Genomic Amplifier Kit is high molecular weight and double-stranded, however a small part of the DNA will be single stranded. Most DNA purification method can be used to generate starting template. In many cases unpurified cell lysates can be directly used as starting material. Typically DNA in the μg range is produced from ng starting material in an overnight incubation at 30°C. |
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| Key Features |
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- Getting unlimited test material from limited sources of DNA material.
- From ng template DNA to μg DNA in an overnight incubation.
- High-quality and representative DNA for genetic analysis, DNA storing and forensic work.
|
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| 5X Amplifier buffer |
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Tris-HCl, pH 7.5, (NH4)2S04, MgCl2, DTT and dNTPs. |
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| Genomic Control DNA |
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Human genomic DNA 10 ng/ml |
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| Primer-mix |
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Hexamers primer-mix for random annealing at 30°C |
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|
| Amplifier Polymerase |
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Enzyme is supplied in 50 mM Tris-HCl pH 7.5, 100 mM KCl, 0.1 mM EDTA, 1 mM DTT, 0.5% Tween 20, 0.5% NP40, 50% glycerol. |
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| Quality Control |
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10 ng of Human genomic DNA will produce between 2-5 ug of DNA. The quality of DNA is judged visually from running the DNA on agarose gel electrophoreses. The quality of the DNA is analysed by Real Time PCR using different primer sets specific for loci on different chromosomes. |
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| Important notes: |
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- 1 ng (350 copies of human genomic DNA) of DNA is required for efficient representative amplification.
- The AmpliQ Genomic Amplifier Kit is extremely sensitive, very small amounts of any input DNA will be efficiently amplified, it is therefore important to use clean implements and containers.
- It is recommended to use as small volume template DNA as possible (1-2 μL) since contaminants within the sample can inhibit the reaction (i.e. SDS, EDTA, hemoglobin, high salt).
- It is recommended to use as intact DNA as possible, since nicked, “old” or restriction enzyme digested DNA will perform poorly in the reaction.
- In absence of template DNA, an amplified non-specific product will likely appear (hexamer primer amplification), however this product will not influence in later applications.
- It should be tested if the various templates can be applied directly or a DNA purification step has to be performed.
- Some applications are sensitive to residual components of the reaction or carry-over from the starting sample. The need for purifying DNA for downstream applications is best determined empirically.
- For some application an extra 3 minutes denaturation step of the template DNA can help to generate more yield.
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| Suggested Protocol for the AmpliQ Genomic Amplifier Kit. |
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This protocol serves as a guideline for DNA amplification. Optimal reaction conditions such as template pre-purification and amount of template DNA may vary and must be individually determined.
- Thaw 10X Amplifier buffer, Genomic Control DNA and Primer-mix solutions. It is important to mix the solutions completely before use to avoid localized concentrations of salts.
- Prepare a master mix according to Table 1.
- Use 1 μL of control DNA (10 ng/μL) and run this reaction parallel as a positive control.
Table 1. Reaction components (master mix and template DNA)
| Component |
Vol./reaction |
Final Conc. |
| 5X Amplifier buffer |
4 μL |
1X |
| Primer-mix |
2 μL |
1X |
| Template DNA |
Variable |
ng range |
| Distilled water |
Variable |
1X |
| Amplifier polymerase |
1 μL |
------- |
| Total volume |
20 μL |
------- |
- Mix the master mix thoroughly and incubate at
30°C overnight (8 – 18 hours). A thermal cycler
can be used as an incubator.
- Heat inactivate the amplifier mix by incubating at
65°C for 10 minutes.
- The tube should now contain amplified DNA that
can be used for many applications directly or after
a purification step.
To verify the amplification run a small part of the
sample on an agarose gel as illustrated in figure 1.
|
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 |
| Figure 1. Amplification of Human genomic DNA with and without heat denaturation. |
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Under standard amplification conditions as described in this datasheet human genomic DNA was amplified overnight using the AmpliQ Genomic Amplifier Kit.
| Lane 1 |
1 ng human genomic DNA with a 95°C template denaturation step. |
| Lane 2 |
1 ng human genomic DNA without a 95°C template denaturation step. |
| Lane 3 |
10 ng human genomic DNA with a 95°C template denaturation step. |
| Lane 4 |
10 ng human genomic DNA without a 95°C template denaturation step. |
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| Quantification of Amplified DNA products |
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The amount of amplified product can be determined using standard UV absorption (OD260). However since the present of polymerase, dNTP and primers will generate inaccurate results, it is recommended to purify the DNA product before using UV absorption (OD260) method. Standard ethanol precipitation purification is sufficient to solve this issue. |
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| Related Products |
| |
| Description |
Cat. No. |
Taq DNA Polymerase (500 Units)
with 10X Ammonium Reaction Buffer
with 10X Standard Reaction Buffer |
110303 |
Taq DNA Polymerase (500 Units)
with 10X Combination Buffer |
110403 |
Taq DNA Polymerase (500 Units)
with 10X Mg++ Free Ammonium Buffer |
110503 |
Taq DNA Polymerase 2.0X Master Mix (100 Reac)
with 2.0 mM MgCl2 |
150301 |
Taq DNA Polymerase 2,0X MaMi RED (100 Reac)
with 1.5 mM MgCl2 |
180301 |
Taq DNA Polymerase 2.0X MaMi RED (100 Reac)
with 2.0 mM MgCl2 |
190301 |
| AccuPOL DNA Polymerase (500 Units) |
210303 |
TEMPase Hot Start DNA Polymerase (500Units)
with 10X TEMPase Buffer I
with 10X TEMPase Buffer II |
220303 |
| UniPOL –Long Range PCR (100 Reac) |
270701 |
| Rapid Ligation Kit (50 React) |
750300 |
| RT-PCR One Tube (100 Reac) |
740301 |
TEMPase Hot Start 2X Master Mix
with TEMPase Buffer I (100 Reac) |
230301 |
TEMPase Hot Start 2X Master Mix
with TEMPase Buffer II (100 Reac) |
230701 |
dNTP Mix (2 x 500μl)
(12.5 mM of each dA, dC, dG and dT) |
501004 |
dNTP Mix, (2 x 500 μl)
(10 mM of each dA, dC, dG and dT), |
502004 |
GC5 Value Efficiency, 108 Cfu/μg pUC19
Chemically Competent Cells, (10x 200μl) |
812010 |
GC5 High Efficiency, 109 Cfu/μg pUC19
Chemically Competent Cells, (10x 50μl) |
805010 |
GC5 High Efficiency, 109 Cfu/μg pUC19
Chemically Competent Cells, (5x 200μl) |
802005 |
SuperPath GC10, 1010 Cfu/μg pUC19
ElectroCompetent Cells, (5x 80μl) |
830805 |
| SOC Medium, 10x 10mL |
800000 |
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Tween 20® is a registered trademark of ICI Americas, Inc. |
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NOTICE
In certain countries, patents cover the PCR process. This product is intended for researchers having a license to perform PCR or those not required to obtain a license. |
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