08 Fungal gDNA Kit

8/2/2019 08 Fungal gDNA Kit

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W e m a k e D N A s p e a k

Services

Xcelris Labs Ltd.

Old Premchand Nagar Road, Opp. Satyagrah Chhavani, Bodakdev,Ahmedabad 380054, India. Tel: +91-79-66197777/ 66311114, Fax: +91-79-66309341www.xcelrisgenomics.comW e m a k e D N A s p e a k

Cat No: XG241

XcelGen

Ver.0.0/11

Fungal gDN

User Gu



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Fungal gDNA Kit

Table of Contents

Introduction

Overview

Storage and Stability

Kit Contents

Before Starting

Fungal gDNA Miniprep Protocol

A.Dry Specimens

B.Fresh/Frozen Specimens

Vacuum/Spin Protocol

Trouble Shooting Guide

Limited Use and Warranty

Fungal gDNA Kit

Introduction Fungal gDNA Miniprep Kits allow rapid and reli

DNA from a wide variety of Fungal species and

to 50 mg dry tissue) can be processed in less

reversible nucleic acid-binding properties ofversatility of spin column technology to elimina

and enzyme inhibitors from fungal tissue lyrestriction digestion, and hybridization techniq

reducing plastic waste and hands-on time to a

parallel.

OverviewIf using the Fungal gDNA Miniprep Kit for t

become familiar with the procedures before

disrupted and then lysed in a specially formula

polysaccharides, and cellular debris are subseare then adjusted and the sample i s applied to

remove trace contaminants such as residual po

water or low ionic strength buer. Puried D

applications without the need for fur ther puri

Storage and StabilityAll components of the Fungal gDNA Miniprep

date of purchase when stored at 22ºC-25ºC. Du

conditions, precipitates may form in Buer FG 3

warming the solution at 37ºC.

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Fungal gDNA Kit

Before StartingPrepare all components and get all necessary materials ready by examining this

instruction booklet and become familiar with each steps.

Important:Pre-heat FG1 and Elution Buer to 65ºC.

Dilute Wash Buer Concentrate with ethanol as follows and store at room temperature.

Add 8 ml (XG2416-00) or 60 ml (XG2416-01) or 96 ml (XG2416-02) absolute (96%-100%)

ethanol to each bottle.

Choose the most appropriate protocol to follow. Procedures are described for each of

dried and fresh (or frozen) specimens.

A. Dry Specimens : For processing 50 mg powdered tissue. Yield is sucient for

several tracks on Southern assay.

B. Fresh or Frozen : For processing 100 mg fresh (or frozen) powdered tissue.

Yield is similar to A.

Fungal gDNA Kit

Fungal gDNA Miniprep Protocol

A. Dry SpecimensMaterials supplied by users: Centrifuge capable of at least 10,000 x g

Nuclease-free 1.5 ml or 2.0 ml microfuge tub

Waterbath equilibrated to 65ºC

Equilibrate sterile ddHO water at 65ºC

Absolute (96%-100%) ethanol

Paper towels

This is the most robust method for isolation of to

genomic) DNA. Yields are usually sucient for severa

Drying allows storage of eld specimens for prol

Samples can be dried overnight i n a 45 ºC oven, pow

prepare dried samples place ~50 mg of dried tissue in

for processing of >50 mg tissue) tube and grind usi

work well. For critical work such as PCR and cloning, p

in a dilute bleach solution immediately after use

autoclaved several times. For standard Southern an

times to grind multiple tissue samples by rinsing

between samples. A ne powder will ensure optima

four to six tubes until Step 2 before starting another

To 10-50 mg powdered dry tissue add 500 l Bue

column at 10,000 x g for 2 min.

Carefully aspirate or decant the supernatant and d

pellet. Invert the microfuge tube on a paper towel f

not necessary to dry the pellet.

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Kit Contents

Product XG2416-00 XG2416-01 XG2416-02

DNA Columns 5 50 250

2 ml Collection Tubes 10 100 500

Buer P1/PB 3 ml 30 ml 150 ml

Buer P2/FG1 3.5 ml 35 ml 175 ml

Buer P3/FG2 1.0 ml 10 ml 50 ml

RNase A 30 l 270 l 1.4 ml

DNA Wash Buer 2 ml 15 ml 3 x 24 ml

Elution Buer 1.5 ml 15 ml 50 ml

User Menu 1 1 1



Fungal gDNA Kit

Adding 600 l Buer FG 1. Vortex vigorously to mix. Make sure to disperse all clumps.

Note: Process in sets of four to six tubes: grind, add Buer FG1, then proceed to Step 2 before starting

another set. Do not exceed 50 mg dried tissue.

Incubate at 65ºC for 30 min. Mix sample twice during incubation by inverting tube. Optional: If

necessary, add 5 l of RNase A into the lysate before incubation to remove the RNA.

Add 600l chloroform/Isoamyl alcohol (24:1) and vortex to mix. Centrifuge at 10,000 x g for 10

min.

Carefully aspirate 300l supernatant to a new 1.5 ml microfuge tube making sure not to disturb the

pellet or transfer any debris.

Adding 150l Buer FG 2 followed by 300l absolute ethanol and vortex to obtain ahomogeneous

mixture. A precipitate may form upon addition of

ethanol; it will not interfere with DNA isolation.

Apply the entire sample (including any precipitate that may have formed) to a DNA column placed

in a 2 ml collection tube (supplied) .Centrifuge the column at 10,000 x g for 1 min to bind DNA.Discard both the 2 ml collection tube and the ow-through liquid.

Transfer column to a second collection tube and wash by adding 650 l DNA Wash Buer diluted

with absolute (96%-100%) ethanol. Centrifuge at 10,000 x g for 1 min and discard the ow-through

liquid.

Note: Wash Buer Concentrate must be diluted with absolute (96%-100%) ethanol prior to use. Follow

directions on label.

Repeat wash step with an additional 650 l DNA Wash Buer. Centrifuge at 10,000 x g for 1 min.

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Fungal gDNA Kit

Centrifuge empty column 2 min at maximum spe

residual ethanol that may otherwise be eluted

applications.

Transfer column to a clean 1.5 ml tube. Apply 100

pre-warmed to 65ºC and incubate at room temperafor 3 to 5 min to elute DNA. Smaller volumes will si

give lower yields. Use of more than 200 l of buer

Repeat Step 9 with an additional 100 l of Elution B

1.5 ml tube to maintain a higher DNA concentratio

Note: To increase DNA concentration, add elution

5 min before elution.

Total DNA yields vary depending on type and quan

a A260/A280 ratio of 1.7-1.9 can be isolated using 5

B. Fresh or Frozen Specimens

Materials to be provided by user:Microcentrifuge capable of 10,000 x g

Nuclease-free microfuge tubes

Waterbath equilibrated to 65ºC

Equilibrate sterile ddHO water at 65 ºC

Absolute (96%-100%) ethanol

Liquid nitrogen for freezing/disrupting sam

Paper towels

Note: Use extreme caution when handling liquid ni

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Fungal gDNA Kit

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This protocol is suitable for most fresh or frozen tissue samples, allowing ecient recovery of

DNA. However, due to the tremendous variation in water and polysaccharide content of various

fungi, sample size should be limited to 200 mg. Best results are obtained with young leaves or

needles. The method isolates sucient DNA for several tracks on a standard Southern assay.

To prepare samples, collect tissue in a 1.5 ml or 2 ml microfuge tube and freeze by dipping in

liquid nitrogen with a pair of tweezers to ll the tube. Grind the tissue using disposable Kontes

pellet pestles. Alternatively, one can allow liquid nitrogen to evaporate and then store samples

at -70ºC for later use. For critical work such as PCR and cloning, pestles are best used a single

time then soaked in a dilute bleach solution immediately after use until clean. Disposable

pestles may be autoclaved several times. For standard Southern analysis, the same pestle can

be reused several times to grind multiple tissue samples by rinsing with ethanol and carefully

wiping the surfaces clean between samples.

Collect ground fungal tissue (start with 100 mg) in a microfuge tube and immediately add 500

l Buer PB,Vortex vigorously to mix. Centrifuge the column at 10,000 x g for 2 min.

Carefully aspirate or decant the supernatant and discard making sure not to dislodge the DNA

pellet. Invert the microfuge tube on a paper towel for 1 min to allow residual liquid to drain. Itis not necessary to dry the pellet.

Adding 500 l Buer FG 1. Vortex vigorously. Make sure to disperse all clumps. DNA cannot be

eectively extracted from clumped tissue.

Note: Process in sets of four to six tubes: ll all tubes with liquid nitrogen, grind, add Buer FG1, then

proceed to Step 4 before starting another set. As a starting point, use 100 mg tissue per tube; if yield

and purity are satisfactory increase to 200 mg.

Incubate at 65 ºC for 15 min. Mix sample twice during incubation by inverting tube.

Optional: If necessary, add 5 l of RNase A into the lysate before incubation to remove the RNA.

Fungal gDNA Kit

Add 800 l chloroform/Isoamyl alcohol (24:1) and v

Centrifuge at 10,000 x g for 5 min.

Carefully aspirate 300l supernatant to a new 1.5 m

pellet or transfer any debri.

Adding 150l Buer FG 2 followed by 300l absolu

mixture. A precipitate may form upon addition of e

Transfer column to a second collection tube and w

with absolute ethanol). Centrifuge at 10,000 x g for

Note: Wash Buer Concentrate must be diluted w

directions on label.

Repeat wash step with an additional 650 l DNA W

Discard ow-through and use a new 2 ml collection

Centrifuge empty column 2 min at maximum speedethanol that may otherwise be eluted with DNA an

Transfer column to a clean 1.5 ml tube. Apply 10

pre-warmed to 65ºC and incubate at room tempera

3 to 5 min to elute DNA. Smaller volumes will sign

lower yields. Use of more than 200 l of buer for e

Repeat Step 9 with an additional 100 l of Elution B

ml tube to maintain a higher DNA concentration in

Tip: To increase DNA concentration, add Elution Bu

5 min before elution.

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Fungal gDNA Kit

Problem Possible reason S

Clogged well Carry-over of debris. F

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Trouble Shooting Guide

DNA pellet not completely I

dissolved before applying d

sample to column. a

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Sample too viscous. I

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DNA remains bound to I

column. i

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Ethanol carry-over F

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Low DNA Incomplete disruption of F

yield starting material. a

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Poor lysis of tissue. D

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Problems in Salt carry-over. D

downstream t

applications

Fungal gDNA Kit

Note: To increase DNA concentration, add elution buer and incubate the column at 60ºC - 65ºC for 5

min before elution.

Total DNA yields vary depending on type and quantity of sample. Typically, 10-50 g DNA with a

A260/A280 ratio of 1.7-1.9 can be isolated using 50 mg dried tissue.

Vacuum/Spin Protocol

Note: Please read through previous sections of this manual before using this protocol.

Prepare wet or dry samples by following the standard Protocol in previous sections until loading

DNA/FG3/Ethanol mixture to DNA column.

Prepare the vacuum manifold according to manufacturer’s instructions and connect the V-Spin

column to the manifold.

Load the DNA/FG3/Ethanol solution to the column.

Switch on vacuum source to draw the sample through the column and turn o the vacuum.

Wash the column by adding 650 l DNA wash buer. Draw the wash buer through the column by

turning on the vacuum source. Repeat this step with another 650 l DNA wash buer.

Assemble the column into a 2 ml collection tube and transfer the column to a micro centrifuge. Spin

1 min to dry the column.

Place the column in a clean 1.5 ml microcentrifuge tube and add 100 l DNA Elution Buer or water.

Stand for 1-2 min and centrifuge 1 minute to elute DNA.

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DNA washed o. D

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Fungal gDNA Kit

Limited Use and Warranty

This product is intended for in vitro research use only. Not for use in human.

This product is warranted to perform as described in its labeling and in XcelGen’s literature when

used in accordance with instructions. No other warranties of any kind, express or implied,

including, without limitation, implied warranties of merchantability or tness for a particular

purpose, are provided by XcelGen. XcelGen’s sole obligation and purchaser’s exclusive remedy for

breach of this warranty shall be, at the option of XcelGen, to replace the products, XcelGen shall

have no liability for any direct, indirect, consequential, or incidental damage arising out of the use,

the results of use, or the inability to use it product.

For technology support or learn more product information, please visit our website at

www.xcelrisgenomics.com

08 Fungal gDNA Kit

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