کیت استخراج RNA از گیاه|Plant RNA isolation kit

کیت استخراج RNA از گیاه|Plant RNA isolation kit

PHYTORENA

Plant RNA Isolation Kit

Cat. No:

FPKT034.0025

FPKT034.0050

FPKT034.0100

Contents:

Kit storage:

 This kit should be stored at room temperature.

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

Additional Equipment and Reagent required

• Make sure everything is RNase-free when handling RNA.
• Absolute ethanol
• ethanol 70%
• Chloroform
• Standard tabletop microcentrifuge capable of 13,000 x g centrifugal force
• Microcentrifuge tubes, 1.5 ml, sterile

Application

The High Pure RNA Isolation Kit is designed for the purification of total RNA from Plants. RNA is suited for other techniques like northern blotting, RNase protection and primer extension. The procedure is optimized to achieve reliable results within Approximately 1 hour (24 samples simultaneously).

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.

LR Buffer and contain guanidinium hydrochloride which is an irritant.
Do not allow LR 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 LR 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

FPKT034.0025-25 prep

Add 24 ml absolute ethanol to PW Bottle.

FPKT034.0050-50 prep

Add 48 ml absolute ethanol to PW Bottle

FPKT034.0100-100 prep

 Add 48 ml absolute ethanol to each PW 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 RNA yield.

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

Protocol

1. Pipette 800 μl of Plant LR Solution, into 1.5 mL microcentrifuge tube (not provided). Weigh the plant tissue – use 50-100 mg of fresh or frozen tissue; up to 20 mg of lyophilized tissue.

Grind the material using one of the following methods:

1. a) Mortar and Pestle

Place up to 100 mg of plant tissue into liquid nitrogen and grind thoroughly with a mortar and pestle.

1. b) Grinding mill

Place up to 100 mg of tissue into a vial containing stainless steel beads. The vial and beads should be precooled with liquid nitrogen. The setup of the mechanical disruption depends on the tissue type. Immediately transfer the tissue powder into a 1.5 mL microcentrifuge tube containing 800 μl of Plant LR Solution. Vortex for 10-20 s to mix thoroughly Incubate for 15 min at room temperature.

2. Add 200 μl Chloroform and shake it for 10-20 s to mix thoroughly Incubate for 3 min at room temperature.
3. 3. Centrifuge at full speed and 4°C for 12 min.
4. transfer 400 μl of the upper phase into a new 1.5ml tube. Add 400 μl Absolute ethanol and mix well generously.
5. transfer the sample to a High Pure Filter Tube. Centrifuge at full speed for 1 min.
6. Discard the flow through add 700 μl PW Buffer and Centrifuge at full speed for 1 min.
7. (Optional for more pure RNA) Discard the flow through add 500 μl PW Buffer and Centrifuge at full speed for 1 min.
8. Leave the tube assembly in the centrifuge and spin it for 3 min at maximum speed (approximately 14,000 rpm) to remove any residual PW Buffer.
9. Discard the Collection Tube and insert the Filter Tube into a clean, sterile RNase free and DNase free 1.5 ml microcentrifuge tube.
10. Add 35 μl ER Buffer to the upper of the Filter Tube. Centrifuge the tube assembly for 1 min at maximum speed. The microcentrifuge tube now contains the eluted Cell RNA. Either use the eluted RNA directly in RT.PCR or store the eluted RNA at −70°C for later analysis.