i-genomic csi ii dna extraction mini kit (season ii)hong kong tech dragon limited rm 808, topsail...

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Instruction Manual

For purification of genomic and mitochondrial DNA from

- Cigarette butts

- Swabs

- Dried blood spots

- Stains

- Hair (hair root / hair shaft)

- Nail clippings

- Chewing gum

- Blood (1~100 μl)

- Tissues

Cat. No. IP17383 | 50 Columns

Version 1.2

i-genomic CSI II DNA Extraction Mini Kit

(Season II)

www.intronbio.co

m

www.intronbio.com

i-genomic CSI II DNA Extraction Mini Kit(Season II) i-genomic CSI II DNA Extraction Mini Kit(Season II)1 22

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Contents

Kit Contents 3

Storage 3

Quality Control 3

Safety Information 4

Product Warranty and Satisfaction Guarantee 4

Product Use Limitations 4

Technical Assistance 4

Precautions and Safety Information 4

Equipment and Reagents to Be Supplied by User 5

Description 6

Characteristics 6

Column Information 6

Important Points before Starting 7

Recovery of Purified DNA 7

Protocols According to the Sample Groups (7 Protocols) 7

i-genomic CSI Kit (Season II) Procedure 8

Protocol A : Cigarette Butts 10

Protocol B : Swabs 11

Protocol C : Dried Blood Spots 12

Protocol D : Stain, Hair, Nail Clipping 13

Protocol E : Chewing Gum 14

Protocol F : Small Volumes of Blood 15

Protocol G : Small Amount of Tissues 16

Technical Information 17

Troubleshooting Guide 19

Related Products 20

iNtRON Distributors 21


Kit Contents

Label Description Contain

Buffer IP Pre-Lysis Buffer 22 ml

Buffer IG Lysis Buffer 22 ml

Buffer IWA Washing Buffer A 40 ml

Buffer IWB(concentrate) 1 Washing Buffer B 10 ml

Buffer IE 2 Elution Buffer 20 ml

Spin Columns(Violet color O-ring) Inserted into a collection tubes(2.0ml tubes) 50 columns

Collection Tubes(2.0 ml tubes) Additionally supplied. 100 tubes

Binding Carrier3 Enhance binding of DNA(store at 4C) 0.4 ml

RNase A Solution 4 20 mg/ml(store at -20C) 0.2 ml

Proteinase K Solution 5 20 mg/ml(store at -20C) 1.1 ml

1 Buffer IWB is supplied as concentrates. Add 40 ml of ethanol (96-100%) according to the bottle label before use.2 Buffer IE is finally 10 mM Tris-HCl (pH 8.0). You may use your lab’s buffer.3 The Binding Carrier should be stored at 4°C upon arrival.4 Store at -20°C. The RNase A solution is completely free of DNase activity. 5 Store at -20°C. After thawing, freshly use. We recommend to aliquot to small volume of Proteinase K.

Storage

We recommend that all component of i-genomic CSI II DNA Extraction Mini Kit(Season II) should bestored at room temperature. However, the two components should be stored separately. Proteinase Kand RNase A should be stored at -20 °C and Binding Carrier should be stored at 4 °C after receiving(Binding carrier will be delivered at frozen condition.)

Quality Control

As iNtRON quality control program, the performance of iNtRON’s products are monitored routinely on alot-to-lot basis. The genomic DNA yield of i-genomic series Genomic DNA Mini Kit is tested bypreparing various sample and assaying the genomic DNA yield spectrophotometrically. The quality ofisolated genomic DNA is checked by restriction digestion, PCR, agarose gel electrophoresis, andspectrophotometry. The i-genomic CSI II DNA Mini Kit (Season II) is tested to ensure the absence ofDNase contamination. All buffers are each incubated with 1 mg pUC18 DNA for 6 hours at 37oC. TheDNA is then visualized by electrophoresis on an agarose gel and compared to a positive control todetermine if any linear or nicked plasmid DNA is visible.

Related Products

Product Name Cat. No.

MaximeTM PCR PreMix (i-Taq)

MaximeTM PCR PreMix (i-StarTaq)

MaximeTM PCR PreMix (i-pfu)

MaximeTM PCR PreMix (i-MAXII)

i-TaqTM plus DNA polymerase

i-genomic CTB DNA Extraction Mini Kit

i-genomic Blood DNA Extraction Mini Kit

i-genomic Stool DNA Extraction Mini Kit

i-genomic Soil DNA Extraction Mini Kit

100bp Ladder Molecular Weight DNA Marker

1 kb Ladder Molecular Weight DNA Marker

PCR Starter Kit (MS-1 Type)

PCR Starter Kit (MS-2 Type)

Agarose

RedSafeTM Nucleic Acid Staining Solution

25025 / 25026

25165 / 25167

25185

25265

25151

17341

17351

17451

17421

24012

24022

25141

25142

32032

21141


Troubleshooting Guide

Problem Possible Cause Recommendation

Low flow rate

in column

Clogged Spin Column by

particulate material

• Completely perform the Disruption & Homogenization step.

• Increase the incubation time at 65C in Lysis step.

High viscosity of Lysate

Reagents correctly

• Reduce the amounts of starting material.


Problem in centrifugation • Check your centrifuge, and then speed up or increase the

centrifugation time.

Low DNA

yield

Inadequate lysis • Reduce the amounts of starting material.


Error in DNA binding • After adding ethanol in DNA Binding step, please mix well by

gently inverting.

• Check that the amount of ethanol is added correctly to the

supernatant

Incorrect Washing step • Check again that the amount of ethanol is added correctly to

Buffer IWB.

• When store Buffer IWB, always keep a lid shut tightly without

evaporation

Insufficient DNA elution • Increase the volume of Buffer IE or water to 100 μl

• Increase the incubation time on the column to 5 ~ 10 min at room

temperature prior to centrifugation.

• Reload the elute, and then incubation for 1 min at room

temperature prior to centrifugation

Binding Carrier was not

added to sample mixture

• Repeat the purification procedure with new samples.

Problems in

downstream

experiments

Ethanol contamination • Ensure that during Buffer IWB, the column membrane should be

dried completely. Please centrifuge at full speed for 5 ~ 10 min to

dry the membrane.

• During Buffer IWB, after centrifugation, remove carefully the Spin

Column from the Collection Tubes without contacting with the

flow-through. This careless contact will result in contamination of

ethanol

Salt contamination • Wash again the Spin Column with Buffer IWB

• Store Buffer IWA and IWB at room temperature (15 ~ 20C).

Amount of DNA used in

experiments

• Optimize the amount of DNA used in your downstream

experiments

Safety Information

When working with chemicals, always should wear a suitable lab coat, disposable gloves, andprotective goggles. For more information, please request the appropriate material safety data sheets(MSDS). Do not add bleach or acidic solutions directly to the waste. Buffer IG and Buffer IWA containsa chaotropic salts, which can form highly reactive compounds when combined with bleach. If liquidcontaining this buffer is spilt, clean with suitable laboratory detergent and water.

Product Warranty and Satisfaction Guarantee

All products undergo extensive quality control test and are warranted to perform as described whenused correctly. Immediately any problems should be reported. Satisfaction guarantee is conditionalupon the customer providing full details of the problem to iNtRON within 60 days, and returning theproduct to iNtRON for examination.

Product Use Limitations

i-genomic CSI II DNA Extraction Mini Kit(Season II) is intended for research use only. Prior to using itfor other purposes, the user must validate the system in compliance with the applicable law, directives,and regulations.i-genomic CSI II DNA Extraction Mini Kit(Season II) is developed, designed, and sold for researchpurpose only. They are not to be used for human or animal diagnosis of diseases. Do not use internallyor externally in humans or animals. Be careful in the handling of the products.

Technical AssistanceAt iNtRON we pride ourselves on the quality and availability of our technical support. Our TechnicalService Departments are staffed by experienced scientists with extensive practical and theoreticalexpertise in molecular biology and the use of iNtRON products. If you have any questions orexperience any difficulties regarding the i-genomic CSI II DNA Extraction Mini Kit(Season II) or iNtRONproducts in general, please do not hesitate to contact us.iNtRON customers are a major source of information regarding advanced or specialized uses of ourproducts. This information is helpful to other scientists as well as to the researchers at iNtRON. Wetherefore encourage you to contact us if you have any suggestions about product performance or newapplications and techniques.For technical assistance and more information please call one of the iNtRON Technical ServiceDepartments or local distributors.

Precautions and Safety Information

All chemicals should be considered as potentially hazardous. When working with chemicals, alwayswear a suitable lab coat and disposable glove. Some buffer contain the chaotrophic salt which may bean irritant and carcinogen, so appropriate safety apparel such as gloves and eye protection should beworn. If a spill of the buffers occurs, clean with a suitable laboratory detergent and water.If the liquid spill contains potentially infectious agents, clean the affected area first with laboratorydetergent and water, then with a suitable laboratory disinfectant. Only persons trained in laboratorytechniques and familiar with the principles of good laboratory practice should handle these products.

CAUTION: DO NOT add bleach or acidic solutions directly to the sample-preparation waste.


Equipment and Reagents to be Supplied by User

The i-genomic CSI II DNA Extraction Mini Kit(Season II) provides almost all reagents for extractingDNA. However, you should prepare some equipments and reagents as follows for a fast and easyextraction. When working with chemicals, always wear a suitable lab coat, disposable gloves, andprotective goggles.

The i-genomic CSI II DNA Extraction Mini Kit(Season II) provides almost

• Ethanol (96 - 100%)

• Micro-pipettes and pipette tips

• Microcentrifuge

• Water bath or heating block

• Vortex mixer

• Micro centrifuge tubes (1.5 ml)

• 1M DTT

• Other general lab equipments

Technical Information

Efficiency Test of DNA Extraction with competitor’sIn order to estimate DNA extraction efficiency from few amount of sample, PCR test and qPCR test were performed

with competitor’s. i-genomic CSI II DNA Extraction Mini Kit(Season II) shows an improved efficiency of genomic

DNA extraction from small amount of sample comparing with competitor’s product.

Fig. 2. Genotyping : Short tandem repeat (STR) analysisShort tandem repeat (STR) analysis of DNA isolated from glass frame swab, using the i-genomic CSI II DNA Extraction Mini Kit

(Season II) procedure. The extracted gDNA was used PCR template using PowerPlex16 system (Promega), the amplified PCR

product was analyzed on an ABI PRISM 3100 DNA Sequencer. The quality of extraction DNA was enough to analyze of genotyping.


Technical Information

Efficiency Test of DNA Extraction with competitor’sIn order to estimate DNA extraction efficiency from few amount of sample, PCR test and qPCR test were performed

with competitor’s. i-genomic CSI II DNA Extraction Mini Kit(Season II) shows an improved efficiency of genomic

DNA extraction from small amount of sample comparing with competitor’s product.

Fig. 1. Comparison of efficiency of gDNA extraction by quantitative PCR.

The efficiency was estimated by qPCR amplification of RNase P gene. DNA was eluated in 30 ml Buffer IE and 1 ml

of each eluate was amplified by real-time PCR. The PCR amplification curve of i-genomic CSI II DNA Extraction Mini

Kit(Season II) showed efficient amplification than competitor's. The Ct Values of the kit showed lower than

competitor’s, also the saturation level is little higher than competitor’s.

Panel A; Normal cigarette butts, panel B; Lane M, cigarette butts contaminated with coffee, panel C ; contaminated

cigarette butts with soil, panel D : Swabs, Panel E : Blood spots, panel F : stains, panel G : Gum, panel H : Bone,

panel I : hair shaft

iNtRON

Competitors

A

iNtRON

Competitors

B

iNtRON

Competitors

C

iNtRON

Competitors

iNtRON

Competitors

iNtRON

Competitors

iNtRON

Competitors

iNtRON

CompetitorsiNtRON

Competitors

D E F

G H I

Description

The i-genomic CSI II DNA Extraction Mini Kit(season II) uses well-established technology forpurification of genomic and mitochondrial DNA from small sample volumes or sizes. The kit provides afast and easy way to purify DNA from small amount sample like forensic samples such as swab, driedspots, stains, cigarette butts, small volume of blood, gum, hair and animal tissue. The enhanced lysisefficiency and binding capacity will help to DNA extraction from various sample as maximized efficiency.The procedure is designed to ensure that there is no sample-to-sample cross-contamination and allowssafe handling of potentially infectious samples. After sample lysis, the simple procedure, which is highlysuited for simultaneous processing of multiple samples, yields pure DNA in less than 30 minutes. DNAis eluted in Buffer IE or water and is immediately ready for use in amplification reactions or for storageat -20C. Purified DNA is free of proteins, nucleases, and other impurities.The kit provide 7 kinds of protocols, You can also extract genomic DNAs from various samples byselecting an appropriate protocol. If you need some more information in selecting a protocol, please donot hesitate to contact our Technical Assist Team.

Characteristics

• Isolated high-quality DNA is suitable for many gene expression profiling techniques : Conventional PCR Quantitative PCR Genotyping such as STR analysis

• Advanced GxN technology for rapid and efficient purification of DNA without ethanol precipitation.

Column Information

• The i-genomic CSI II DNA Extraction Mini Kit(Season II) Spin Column

Do not store the Column packs under completely dried conditions. It may be affected to DNA binding capacity.

The Spin Columns are stable for over 2 year under these conditions

Additional Collection Tubes (100 ea) are also supplied for your convenient handling.

1

2

Column membrane1

Spin Column1

Loading Volume

DNA Binding Capacity

Recovery

Elution Volume

Silica-based membrane

Individually, in inserted in a 2.0 ml Collection Tube2

Maximum 800 μl

Maximum 45 μg

85 - 95% depending on the elution volume

Generally, eluted with 30 – 200 μl of elution buffer


Important Points before Starting

• Buffer IWBBuffer IWB is supplied as concentrate. Before using for the first time, be sure to add 40 ml of absoluteethanol(96 - 100%) to obtain a working solution.

• Proteinase K Solution(20 mg/ml)Proteinase K possesses a high specific activity witch remains stable over a wide range oftemperature and pH values with substantially increased activity at higher temperature. Proteinase KSolution shows a milk-white color, since it is supplied as concentrate. After thawing, freshly use. DONOT heat to redissolve. We recommend to aliquot to small volume of Proteinase K Solution

• Preheat a water bath or heating block

• Equilibrate samples to room temperature (15-25 °C).

• Equilibrate Buffer IE or distilled water for elution to room temperature.

• If Buffer IP or Buffer IG contains precipitates, dissolve by heating to 70 °C with gentle agitation.

• CentrifugationAll centrifugation steps are carried out at RT (15 - 25°C)

Recovery of Purified DNA

The yield of DNA isolated from biological samples strongly depends on the amount and source of the starting

material. In addition, DNA size can also vary between 30 kb (in fresh samples) or be below this range in forensic

samples.

If DNA yields are below 1 μg, quantification using a spectrophotometer will be difficult. In this case, we recommend

quantitative amplification methods for determination of yield.

If purifying DNA from very small amounts of sample, such as 1-10 μl blood or forensic samples, we recommend

adding binding carrier to the sample. Be aware that the sample will then contain considerably more binding carrier.

Therefore, when quantifying the purified DNA, avoid quantification methods that are not specific for DNA.

Protocols According to the Sample Groups (7 Protocols)

Samples

Cigarette Butts

Swabs (Blood swab, fingerprint swab etc)

Dried Blood Spots (Blood card punches)

Stain / Hair (hair root, hair shaft) / Nail Clipping

Chewing Gum

Blood (Small amount : 1~100 μl)

Tissues

Protocol Type

Type A Protocol

Type B Protocol

Type C Protocol

Type D Protocol

Type E Protocol

Type F Protocol

Type G Protocol

Protocol G (Small Amount of Tissues)

1. Transfer a tissue sample of less than 10 mg in weight to a 1.5 ml microcentrifuge tube (not provided)..

2. Add 200 μl of Buffer IP, 20 μl of Proteinase K Solution, 3 μl of RNase A and 7.5 μl Binding Carrier into sample

tube and mix vortexing vigorously. Then Incubate the lysate at 65°C for overnight.Note : For small amounts of tissue, lysis is complete in 4–6 h, but best results are achieved after overnight lysis. Vortex the

tube for 10 s every 1-2 hr to improve lysis

3. Briefly centrifuge the 1.5 ml tube to remove drops from the inside of the rid.

4. Add 200 μl of Buffer IG into the lysate, and mix well by gently inverting 5 - 6 times. After mixing, Then Incubate

the lysate at 65°C for 10 min.


6. Add 200 μl of absolute ethanol into the lysate, and mix well by gently inverting 5 - 6 times or by pipetting. DO

NOT vortex. After mixing, Centrifuge at 13,000 rpm for 1 min.Note : This step is an equilibration step for binding genomic DNA to column membrane. It is important to assure proper mixing

after adding the ethanol, until not showing 2-phase which is not mixed. Also, this step conduces to pass efficiently cell lysate

through a column.

7. Carefully apply 600 μl of the supernatant from step 6 to the Spin Column (in a 2 ml Collection Tube) without

wetting the rim, close the cap, and centrifuge at 13,000 rpm for 1 min. Discard the filtrate and place the Spin

Column in a 2 ml Collection Tube (reuse).Note : Close each Spin Column in order to avoid aerosol formation during centrifugation. Do not transfer any solid materials.

8. Repeat by applying up to 100 μl of the remaining supernatant from step 6 to the Spin Column.

9. Place the Spin Column into a new 2.0 ml Collection Tube (additionally supplied), add 700 μl of Buffer IWA to

the Spin Column, and centrifuge for 1 min at 13,000 rpm. Discard the flow-through and reuse the Collection

Tube.

10. Add 700 μl of Buffer IWB to the Spin Column, and centrifuge for 1 min at 13,000 rpm. Discard the flow-through

and place the Column into a new 2.0 ml Collection Tube (additionally supplied), Then again centrifuge for

additionally 1 min to dry the membrane. Discard the flow-through and Collection Tube altogether.Note : It is very important to dry the membrane of the Spin Column since residual ethanol may inhibit subsequent reactions.

Following the centrifugation, remove carefully the Spin Column from the Collection Tube without contacting with the flow-

through, since this will result in carryover of ethanol.

Note : Ensure that 40 ml of absolute ethanol has been added to Buffer IWB.

11. Place the Spin Column into a new 1.5 ml tube (not supplied), and 30 - 100 μl of Buffer IE directly onto the

membrane. Incubate for 1 min at room temperature and then centrifuge for 1 min at 13,000 rpm to elute. Note :

Elution with 30 μl (instead of 50 μl) increases the final DNA concentration, but reduces overall DNA yield conventionally.

Note : A new 1.5 ml tube can be used for the second elution step to prevent dilution of the first eluate. Alternatively, the tube

can be reused for the second elution step to combine the eluates.

Tis

sues


Protocol F (Small Volumes of Blood)

1. Pipet 1-100 μl whole blood into a 1.5 ml microcentrifuge tube (not provided).

2. Add Buffer IP to a final volume of 100 μl.

3. Add 20 μl of Proteinase K, 3 μl of RNase A Solution and 7.5 μl Binding Carrier into sample tube and gently mix.Note : It is possible to add Proteinase K to blood sample that have already been measured into 1.5 ml tube. It is important to

assure proper mixing after adding the Proteinase K and RNase A solution.

Note : The Binding Carrier improves recovery of small amount of DNA. Be sure that Proteinase K solutions are always kept

under freezer (below -10°C).

4. Add 100 μl of Buffer IG into upper sample tube and mix throughly.Note : Avoid any vigorous vortexing because doing so many induce genomic DNA breakage. In order to assure efficient lysis,

it is important that the blood sample and Buffer IG are mixed thoroughly to yield a lysis solution.

5. Incubate the lysate at 65°C for 10 min.Note : For complete lysis, mix 3 or 4 times during incubation by inverting tube. If it lysis perfectly, the red color of lysate

becomes the dark green.



NOT vortex. After mixing, briefly centrifuge the 1.5 ml tube to remove drops from inside of the lid.Note : This step is an equilibration step for binding genomic DNA to column membrane. It is important to assure proper mixing


through a column.

8. Carefully apply the mixture from step 7 to the Spin Column (in a 2 ml Collection Tube) without wetting the rim,

close the cap, and centrifuge at 13,000 rpm for 1 min. Discard the filtrate and place the Spin Column in a new 2

ml Collection Tube (additionally supplied).Note : Close each Spin Column in order to avoid aerosol formation during centrifugation. Do not transfer any solid materials.

9. Add 700 μl of Buffer IWA to the Spin Column, and centrifuge for 1 min at 13,000 rpm. Discard the flow-through

and reuse the Collection Tube.








membrane. Incubate for 1 min at room temperature and then centrifuge for 1 min at 13,000 rpm to elute.Note : Elution with 30 μl (instead of 50 μl) increases the final DNA concentration, but reduces overall DNA yield conventionally.



Sm

all Blo

od

The i-genomic CSI II DNA Extraction Mini Kit

(Season II) Procedure

- Prepare appropriate samples

(refer to protocols)

- Transfer the samples

- Add appropriate volume of Buffer IG (refer to protocols),

20 μl Proteinase K, 3 μl RNase A and 7.5 μl of Binding Carrier

(optional) Some of sample are needed 20 μl of 1M DTT

- Incubate the sample. The incubation time is depended on sample

(refer to protocols)

- Then vortex vigorously

- Add appropriate volume of Buffer IG

- Then vortex vigorously

- Incubate the sample for 10 min.

- Add appropriate volume of 100 % Ethanol

- Then mix thoroughly

- Transfer the whole lysate into the Spin Column and

Centrifuge 13K rpm for 1min.

- Discard the flow-through then place the Spin Column

into a new Collection Tube.

Briefly spin down

Briefly spin down

Briefly spin down

1

2

3

4

5

6

7

8


- Add 700 μl of Buffer IWA into the Spin Column and


- Discard the flow-through then place the Spin Column into a

Collection Tube. (reuse)

- Add 700 μl of Buffer IWB into the Spin Column and


Note: Ensure the 40 ml of EtOH has been added to Buffer IWB

- Discard the flow-through then place the Spin Column into a

new Collection Tube.

- Centrifuge 13K rpm for 1min (Column drying) and discard

flow-though.

- Place the Spin Column into a new 1.5ml micro tube

- Add 30 - 100 μl of Buffer IE and incubate at RT for 1min

- Recover the ready-to -use DNA

Centrifuge 13K rpm for 1min

9

10

11

12

13

The i-genomic CSI II DNA Extraction Mini Kit

(Season II) Procedure

Protocol E (Chewing Gum)

1. Cut up to 30 mg of chewing gum into small pieces and transfer them to a 1.5 ml microcentrifuge tube (not

provided).


tube and mix vortexing vigorously. Then Incubate the lysate at 65°C for 3 hr - overnight.Note : Be sure that Proteinase K solutions are always kept under freezer (below -10°C).

Note : The Binding Carrier improves recovery of small amount of DNA.

Note : Vortex the tube for 10 s every 10 min to improve lysis






NOT vortex. After mixing, Centrifuge at 13,000 rpm for 1 min.Note : This step is an equilibration step for binding genomic DNA to column membrane. It is important to assure proper mixing


through a column.

7. Carefully apply 800 μl of the supernatant from step 6 to the Spin Column (in a 2 ml Collection Tube) without

wetting the rim, close the cap, and centrifuge at 13,000 rpm for 1 min. Discard the filtrate and place the Spin

Column in a 2 ml Collection Tube (reuse).Note : Close each Spin Column in order to avoid aerosol formation during centrifugation. Do not transfer any solid materials.

8. Repeat step 7 by applying up to 100 μl of the remaining supernatant from step 6 to the Spin Column. Discard

the filtrate and place the Spin Column in a new 2 ml Collection Tube (additionally supplied).













Ch

ewin

g g

um


Protocol D (Stain, Hair, Nail Clipping)

1. Prepare sample.

1) Stains (stained with blood, saliva, or semen) : Cut out up to 0.5 cm2 of stained material and then cut it into

smaller pieces. Transfer the pieces to a 2 ml microcentrifuge tube (not provided).

2) Hair root : Cut off a 0.5 - 1 cm piece starting from the hair bulb and transfer it to the 1.5 ml microcentrifuge

tube not provided).

3) Hair shaft : Cut the hair shaft into 0.5 - 1 cm pieces and transfer them to the 1.5 ml microcentrifuge tube

(not provided). Close the lid and mix by pulse-vortexing for 10 s

4) Nail Clipping : Transfer the nail clippings to a 1.5 ml microcentrifuge tube (not provided)

2. Add 300 μl of Buffer IP, 20 μl of Proteinase K Solution, 3 μl of RNase A, 20 μl of 1M DTT (not provided) and 7.5

μl Binding Carrier into sample tube and mix vortexing vigorously. Then Incubate the lysate at 65°C at least 1 hr.Note : In general, hairs are lysed in 1 h. If necessary, increase the incubation time to ensure complete lysis. If using a heating

block or water bath, vortex the tube for 10 s every 10 min to improve lysis.

Note : For larger samples of nail clippings, we recommend overnight incubation at 65°C. Any material that is not lysed during

this incubation step or the incubation in step 4 will be pelleted during centrifugation in step 5.






NOT vortex. After mixing, briefly centrifuge the 1.5 ml tube to remove drops from inside of the lid.

7. Carefully apply 800 μl of the mixture from step 7 to the Spin Column (in a 2 ml Collection Tube) without wetting

the rim, close the cap, and centrifuge at 13,000 rpm for 1 min. Discard the filtrate and place the Spin Column in

a 2 ml Collection Tube (reuse).Note : Close each Spin Column in order to avoid aerosol formation during centrifugation. Do not transfer any solid materials.

8. Repeat step 7 by applying up to 100 μl of the remaining mixture from step 6 to the Spin Column. Discard the

filtrate and place the Spin Column in a new 2 ml Collection Tube (additionally supplied).













Stain

, Hair, N

ail

Protocol A (Cigarette butt)

1. Prepare sample.Note : Air-dry the cigarette butts for at least 2 hr after collection. After sample collection, samples can be kept at room

temperature when processed immediately.

2. Place small pieces of cigarette butts into a 1.5 ml micro-centrifuge tube.Note : Cigarette butts are cut out a 1 cm2 piece of outer paper from the end of the cigarette filter. Cut this piece into 6 smaller

pieces. Transfer the pieces to a 1.5 ml microcentrifuge tube


tube and mix vortexing vigorously.Note : Be sure that Proteinase K solutions are always kept under freezer (below -10°C).

Note : The Binding Carrier improves recovery of small amount of DNA.

4. Incubate the lysate at 65°C for 3 hr - overnight.Note : If using a heating block or water bath, vortex the tube for 10 s every hours to improve lysis.


6. Add 300 μl of Buffer IG into the lysate, and mix well by gently inverting 5 - 6 times.

7. After mixing, incubate the lysate at 65°C for 10 min.





through a column.



a 2 ml Collection Tube (reuse).Note : Close each Spin Column in order to avoid aerosol formation during centrifugation. Do not transfer any filter paper.

11. Repeat step 10 by applying up to 100 μl of the remaining mixture from step 9 to the Spin Column. Discard the

filtrate and place the Spin Column in a new 2 ml Collection Tube (additionally supplied).













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aret

te b

utt


Protocol B (Swabs)

1. Prepare sample.Note : To collect a sample, scrape the swab firmly against the surface of each sample more than 6 times. Air-dry the swab for

at least 2 hr after collection. After sample collection, samples can be kept at room temperature when processed immediately.

If storage is necessary, freeze swab sample at - 20 °C.

2. Place single swab into a 1.5 ml micro-centrifuge tube.Note : Cotton or DACRON swabs are cut from the stick by scissors.


tube and mix vortexing vigorously. Then Incubate the lysate at 65°C at least 1 hr. Note :

Be sure that Proteinase K solutions are always kept under freezer (below -10°C).

Note : The Binding Carrier improves precipitation of small amount of DNA was optimize DNA recovery.








through a column.



a 2 ml Collection Tube (reuse).Note : Close each Spin Column in order to avoid aerosol formation during centrifugation. Do not transfer any solid materials.

9. Repeat step 8 by applying up to 500 - 600 μl of the remaining mixture from step 7 to the Spin Column. Discard

the filtrate and place the Spin Column in a new 2 ml Collection Tube (additionally supplied).










membrane. Incubate for 1 min at room temperature and then centrifuge for 1 min at 13,000 rpm to elute. Note :

Elution with 30 μl (instead of 50 μl) increases the final DNA concentration, but reduces overall DNA yield conventionally.



Sw

ab

Protocol C (Dried Blood Spots)

1. Cut 3 mm (1/8 inch) diameter punches from a dried blood spot with a single-hole paper punch. Place up to 3

blood card punches into a 1.5 ml microcentrifuge tube (not provided).

2. Add 200 μl of Buffer IP, 20 μl of Proteinase K Solution, 3 μl of RNase A, and 7.5 μl Binding Carrier into sample

tube and mix vortexing vigorously. Then Incubate the lysate at 65°C at least 1 hr.Note : If using a heating block or water bath, vortex the tube for 10 s every 10 min to improve lysis.






NOT vortex. After mixing, briefly centrifuge the 1.5 ml tube to remove drops from inside of the lid.



a new 2 ml Collection Tube (additionally supplied).Note : Close each Spin Column in order to avoid aerosol formation during centrifugation. Do not transfer any solid materials.













Dri

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i-genomic csi ii dna extraction mini kit (season ii)hong kong tech dragon limited rm 808, topsail...

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