کیت استخراج DNA از گیاه|Plant Genomic DNA Extraction kit

کیت استخراج DNA از گیاه|Plant Genomic DNA Extraction kit

PHYTOKIA

Plant DNA Isolation Kit

Cat. No:

FPKT022.0025

FPKT022.0050

FPKT022.0100

Contents:

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Kit storage:

This kit should be stored at room temperature.

Proteinase K should be stored at -20 °C

If properly stored, all kit components are stable until the expiration date printed on the label.

After adding absolute ethanol to PP3 and PP4 buffers, Store them at +2 to +8°C

Additional Equipment and Reagent required

• Absolute ethanol
• Standard tabletop microcentrifuge capable of 13,000 x g centrifugal force
• Microcentrifuge tubes, 1.5 ml, sterile

Application

This kit provides a convenient and rapid method to isolate total DNA from fresh, frozen and preserved Plant samples. The purified DNA is of the highest quality and is fully compatible with all downstream applications such as PCR, qPCR, NGS and microarrays since all humic acid substances and other PCR inhibitors are removed during the isolation process.

The procedure is optimized to achieve reliable results within 70 min.

Handling Requirements and Safety Information

All solutions are clear and should not be used when precipitates have formed. Warm the solutions at +15 to +25°C or in a 37°C water bath until the precipitates have dissolved.

PP2 Buffer contain guanidinium hydrochloride which is an irritant.

Do not allow PP1 Buffer and PP2 Buffer to touch your skin, eyes, or mucous membranes. If contact does occur, wash the affected area immediately with large amounts of water.

If you spill the reagent, dilute the spill with water before wiping it up.

Do not use any modified ethanol.

Do not pool reagents from different lot numbers.

Immediately after usage, close all bottles in order to avoid leakage, varying buffer concentrations or buffer conditions.

After first opening store all bottles in an upright position.

Do not allow the PP1 Buffer and PP2 Buffer to mix with sodium hypochlorite found in commercial bleach solutions. This mixture can produce a highly toxic gas.

Wear protective disposable gloves, laboratory coats and eye protection, when handling samples and kit reagents.

Do not contaminate the reagents with bacteria, virus, or nucleases. Use disposable pipets and nuclease free pipet tips only, to remove aliquots from reagent bottles.

preparation procedure:

Working Solution Preparation

FPKT022.0025-25 preps

Add 5 ml absolute ethanol to PP3 Buffer Bottle.

 Add 24 ml absolute ethanol to PP4 Buffer Bottle.

FPKT022.0050-50 preps

Add 10 ml absolute ethanol to PP3 Buffer Bottle.

Add 48 ml absolute ethanol to PP4 Buffer Bottle.

FPKT022.0100-100 preps

Add 20 ml absolute ethanol to PP3 Buffer Bottle.

Add 48 ml absolute ethanol to each PP4 Buffer Bottle.

Please note that the Ethanol concentration of a Washing Buffer may decrease during long term storage resulting in a drop-down of the final DNA yield.

before starting Incubate the Elution buffer at 55 to 65 ° C until the end of the protocol to obtain the maximum yields.

Protocol

1. Sample preparation

Add Fresh or frozen tissue may be finely ground with a mortar and pestle in liquid nitrogen prior to DNA isolation. Work quickly and keep tissue cold to minimize

2.  Transfer the finely ground tissue (100-150 mg) to a 1.5 ml microtube. Add 500 μl PP1 Buffer and 20 μl Proteinase K to the tissue, mix by vortex. Incubate at 60 °C for 60 min.
3. Add 500 μl PP2 Buffer to the sample microtube and Centrifuge at full speed (~18,000 x g) for 5 minutes.
4. Transfer the supernatant into a new 1.5 ml microtube.
5. Add equal volume of Absolute ethanol and mix it by vortexing (10 s)
6. Transfer 800 μl of supernatant from stage 5 into a DNA spin column assembled in a clean collection tube (provided). Centrifuge at 10000 g for 1 min at room temperature.
7. Disconnect the DNA spin column from collection tube and discard the flow through solution. Reconnect the DNA spin column to the collection tube.
8. Add 500 μl PP3 Buffer to DNA spin column and centrifuge at 10000 g for 1 min. Discard flow through.
9. Add 700 μl PP4 Buffer to DNA spin column and centrifuge at 10000 g for 1 min. Discard flow through.
10. Centrifuge the DNA spin column at 10000 g for 1 min to remove residual ethanol.
11. Place the DNA spin column into the clean new microtube. Add 30 μl of pre-warmed Elution Buffer or sterile DDW directly onto column membrane and stand for 3 min. Centrifuge at 10000 g for 1 min.
12. Add another 30 μl pre-warmed Elution Buffer or sterile DDW directly onto column membrane and stand for 3 min. Centrifuge at 10000 g for 1 min.
13. Check 7-10 μl of extracted DNA by 1% agarose gel electrophoresis. Store DNA at -20 ̊C