TOP General Genomic. DNA Purification Kit (mini-prep) For laboratory use only. Cat. No.: TGk1003 (50 prep) TGk (25 prep)

Transcription

1 TOP General Genomic DNA Purification Kit (mini-prep) Cat. No.: TGk1003 (50 prep) TGk (25 prep) For laboratory use only

2 Table of Contents Kit Contents and Storage Kit overview...3 Kit advantages....3 Kit limitations...3 Downstream Applications...3 Extraction procedure..4 Precaution 4 Starting material Materials, Reagents and equipment to be supplied by user....5 DNA extraction from blood, Buffy coat, body fluid, lymphocyte and cultured cells 5 DNA extraction from animal tissues...9 DNA extraction from bacteria and Archeae Typical DNA yield.13 Appendix 1: collection, storage and preparation of starting material s guidelines..15 Appendix2: elution procedures.16 Troubleshooting guide...17 Technical Assistance and Ordering Information...19 Product Warranty 19 References 19 Procedure flowchart.20 1

3 Kit Contents and Storage Shipping, Storage and Stability Top General Genomic DNA Purification kit s all solutions and buffers must be stored at +15 to +25 C. Proteinase K is stable at ambient temperatures but for long storage it s highly recommended that keep the proteinase K solution at 2-8 C. Please consider that storage of other buffers and solution of the kit at +2 to +8 C (refrigerator) or -15 to -25 C (freezer) will case the forming of precipitates in buffers and so will reduce the efficient of DNA extraction significantly. All buffers and solutions of the kit are stable for at least 12 months at room temperature. Kit contents All solutions are clear. If any solution contains a precipitate, do not use it. Instead, warm the solution in a 37 C water bath to dissolve the precipitate. Kit components TL buffer (Tissue lysis buffer) BL Buffer ( lysis/binding buffer) WA buffer (Concentrate) Amounts for 50 test 15 ml 15 ml 15 ml (Wash buffer A) WB solution (Concentrate) 10 ml (Wash buffer B) EB buffer (Elution buffer) Proteinase K (20mg/ml) 10 ml 0.5 ml Spin column 50 2 ml Collection tube 50 * Bring concentrated solutions up to final volume (indicated on the each bottle) with absolute Ethanol. Refer to Before Starting section for more information. 2

4 Kit Overview Top General Genomic DNA Purification kit (Mini-prep) provides a simple and fast method to isolate total genomic DNA from wide range of source materials including whole blood, plasma, serum, Buffy coat, bone marrow, other body fluids, lymphocytes, cultured cells and animal tissues, and bacteria. The isolation protocol and all buffers are optimized to assure a high yield as well as a high purity of purified genomic DNA. All manual work is reduced to a minimum. The resulting DNA is essentially free of RNA, protein, and other enzymatic inhibitors. Samples of interest are first lysed and homogenized in the presence of lysis buffer, then ethanol is added to provide appropriate binding conditions to the silica membrane, and the sample is applied to a spin column, where the genomic DNA binds to the membrane and contaminants are efficiently washed away. High-quality DNA is then eluted in μl of elution buffer. The purified DNA is ready to use for applications such as PCR and Real time PCR, southern blotting and etc. Kit Advantages High pure DNA yield: Purified DNA is of the highest quality and quantity proper enough for any downstream application. User friendly: the kit provides all reagents and protocols needed for isolation of high pure DNA from various source samples including; whole blood, body fluids and cultured cells. Time saving: Required time for preparation of genomic DNA is approx min per sample. The kit allows processing of 10 samples in less than 50 min. Increased lab safety: No need for phenol/ chloroform and other hazardous chemicals. Kit Limitations: Top General Genomic DNA purification kit is intended for molecular biology applications. This product has been developed just for research purposes. Downstream Applications The purified DNA can be used in a wide range of downstream applications, including: PCR and quantitative real-time PCR Southern blotting SNP genotyping Pharmacogenomic studies 3

5 Extraction Procedure Precaution All human and animal sourced material and all resulting waste should be considered as potentially infectious. Ensure that a suitable lab coat, disposable gloves and protective goggles are worn and standard safety precautions are followed when working with chemicals. Buffer BL and Buffer WA contain a guanidine salt and are therefore not compatible with disinfecting reagents containing bleach. Do not add bleach or acidic solutions directly to solutions or sample preparation waste that contains guanidinium salts, as reactive compounds and toxic gases are formed. WA and WB are containing ethanol and considered flammable Use appropriate precautions when using these chemicals. Starting material Use the starting material according the table 1: Table 1, Source sample Amount Mammals blood, Plasma, serum 200 µl Buffy coats 200 µl Fish, bird and frog blood* 5 to 10 µl Diploid cells Up to Animal tissues Up to 25 mg bacteria 2 x 10 9 *Bird blood contains nucleated erythrocytes, giving higher DNA yields than mammalian blood. * When isolating DNA from spleen, 10 mg samples should be used Note: Do not use higher volume of samples, since these will overload the effective materials of buffers or solutions and reduce the yield. 4

6 Materials, Reagents and equipment to be supplied by user Refer to the list below for additional reagents and equipment required Absolute ethanol PBS, ph 7.2 (50 mm potassium phosphate, 150 mm NaCl) Enzymatic lysis buffer for pretreatment of gram positive bacteria: (20 mm Tris Cl, ph mM Na-EDTA, 1.2 % Triton X-100 and immediately before use add Lysozyme to 20 mg/ml. RNase A (100 mg/ml) 60 C and 70 C water bath or heat block High speed microcentrifuge Microcentrifuge tubes, ml, sterile DNA extraction from blood, plasma, serum, buffy coat, body fluid, lymphocytes and cultured cell samples. Before Starting Please read the Appendix 1; Collection, storage and preparation of starting material s guidelines. Perform all steps, including centrifugations, at room temperature. BL buffer may form a precipitate upon storage if necessary warm to 56 C until the precipitate has fully dissolved. Mix BL buffer thoroughly by shaking before use. Buffer WA and buffer WB are supplied as concentrates. Before using for the first time add 20 ml of ethanol (96-100%) to WA and 44 ml of ethanol (96-100%) to WB to obtain a working solution. 200 μl of whole blood yields 3-7 μg of DNA according the sample. White Blood cell precipitation or Preparation of Buffy coat (page 13) is recommended if a higher yield is required. When preparing DNA from buffy coat or lymphocytes, full-speed centrifugation is recommended to avoid clogging. 5

7 Extraction recipe For blood samples 1.a) for direct DNA extraction from whole blood and related tissues: Pipette 200 µl of mammal s blood, plasma, serum, body fluids and buffy coats into a 1.5 or 2 ml microcentrifuge tube and add 10 µl of proteinase K to the samples and mix well by Vortexing or pipetting. For frog, bird and fish samples use µl of whole blood as starting material. Add 10 µl of proteinase K and Adjust the volume of the whole sample to 200 µl using PBS buffer. Continue with step 2. Note: for mammal s sample volumes less than 200 µl, add 10 µl of proteinase K and adjust the volume of the sample to 200 µl with PBS. 1.b) Recommended if more DNA from blood is preferred: for DNA extraction from white blood cells, Lyse red blood cells from µl Blood, plasma, serum by any desired RBC lysis Buffer. Note that the number of cells is not more than cells. Follow the instructions for cultured cells to extract DNA For cultured cells 1. Centrifuge 1-5 x 10 6 cells for 5 min at 3000 rpm. Resuspend the pellet in 200μl PBS. Add 10 μl of proteinase K, mix well by pipetting or vortexing and continue with step2. When using a frozen cell pellet, allow cells to thaw before adding PBS. Do not use more than cells for DNA extraction with this protocol. For cell lines with a high degree of ploidy (e.g., HeLa cells), it is recommended to use less than the maximum number of cells. Optional: RNA may inhibit some downstream enzymatic reactions, but not PCR. If RNA-free genomic DNA is required, 4 μl of an RNase A stock solution (100 mg/ml) should be added to the sample before addition of Buffer BL. 2. Add 200 µl buffer BL. Mix immediately and completely by pipetting. Note: Avoid any vigorous vortexing to avoid of genomic DNA breakage. In order to assure efficient lysis, it is important that the blood sample and Buffer BL are mixed thoroughly to yield a lysis solution. For samples with larger volume of 200 µl increase the amount BL buffer proportionally. 3. Incubate the lysate at 70 C for 10 min. DNA yield reaches a maximum after lysis for 10 min at 70 C. Longer incubation times have no effect on yield or quality of the purified DNA. 4. Briefly centrifuge the 1.5 ml micro-centrifuge tube to remove drops from the inside of the lid. 6

8 5. Add 200 µl absolute ethanol (96-100%) to the sample and mix thoroughly by pipetting (at least 10 times) or by gently inverting several times or by pulse vortexing for 15s to homogenize solution until 2 formed separate phases ( ethanol and lysate) are disappeared. Do not vortex vigorously. After mixing briefly centrifuge (short spin) the tube to remove the drops from the lid. Note: mixing of the ethanol and the lysate is very important and essential to prepare good binding conditions of the genomic DNA to the silica column. For larger sample volumes please keep the volume of ethanol/ BL buffer/ starting material in the proportion of 1:1:1, For example for 300 µl starting material use 300 µl of BL buffer and 300 µl of ethanol. 6. Place the spin column (green ring) into a 2 ml collection tube (provided). Pipette the whole of mixture into the mini spin column placed in a 2 ml collection tube without wetting the rim. Close the cap gently and centrifuge at >8000 x g (>11000 rpm) for 1 min. Discard the flow-through *. Note: the maximum capacity of the spin column to load lysis/binding solution is 800 µl. for larger amounts load the sample into the spin column in two steps sequentially. If the lysate has not completely passed through the column after centrifugation, centrifuge again at higher speed until the spin column is empty. It s highly recommended that discard the flow-through using pipettor to avoid the blood splashing or the contamination of the microcentrifuge with blood wastes remained on the rim of the collection tube. 7. Place the mini spin column in the same collection (reuse). Add 500 µl of WA buffer, wait for few seconds and centrifuge at >8000 x g (>11000 rpm) for 1 min. Discard the flow-through. 8. Place the mini spin column in the same collection tube. Add 500 µl of WB buffer and centrifuge at >8000 x g (>11000 rpm) for 1 min, and discard the flow-through. 9. Place the mini spin column in the same tube. Add 500 µl of WB buffer and centrifuge at >16000 x g (>14000 rpm) for 3 min. Discard the flow-through and collection tube. Note: it s important to dry the silica membrane, since residual ethanol may interfere with subsequent reactions. This centrifuge step will remove all ethanol from the membrane. Following the centrifuge step, remove the column carefully to avoid the contact with the flowthrough. If carryover of ethanol occurs, empty the collection tube, place the mini spin column in it, and centrifuge the empty column at >16000 g (>14000 rpm) for 1 min. * - Flow-through is containing buffer BL and therefore is not compatible with bleaches. The buffers BL and WA will form very toxic gases in the contact with bleaches. 7

9 10. Place the mini spin column in a clean 1.5 or 2 ml micro centrifuge tube (not-provided), and pipette 2 x 75 µl of EB buffer directly to the center of silica membrane. Incubate at room temperature for 5 min and centrifuge for 1 min at >11000 rpm. Important: If the DNA concentrations more than 50ng/µl are needed for downstream applications use μl of buffer EB. The concentration of purified DNA from 200 μl of whole blood from healthy human is about 34 ng/μl in 150 μl of buffer EB. Elution of DNA in 75µl buffer EB will increase the concentration of DNA up to 72ng/μl but the total amount of recovered DNA will be reduced down to 90% of total DNA. Elution of DNA in 2x75 μl will increase the total amount of released DNA in competition with 1x75 μl or 1x150 μl elution buffer (see the table 3 for more information). For samples containing less than 1 μg of DNA, elution in μl Buffer EB or water is recommended. Eluting with 2 x 75 μl instead of 1 x 150 μl will increase elution efficiency up to 15%. For long-term storage of DNA, eluting in Buffer EB or TE (Tris-Cl 10mM, ph=8, EDTA 1mM) and storing at 20 C is recommended, since DNA stored in water is subject to acid hydrolysis Important: For more information about elution procedure please refer to the appendix 2: Elution procedures. Table3. The average purified DNA yield according the volume of EB buffer. DNA extraction from animal tissues: Before Starting Sample volume (healthy human blood) Average yield Elution volume concentration 200µl 5.1 µg 150 µl 34 ng/μl 200 µl 6 µg 2x75 µl 40 ng/μl 200 µl 5.4 µg 1x75 µl 72 ng/μl Please read the Appendix 1; Collection, storage and preparation of starting material s guidelines. 8

10 Perform all steps, including centrifugations, at room temperature. BL buffer may form a precipitate upon storage if necessary warm to 56 C until the precipitate has fully dissolved. Mix BL buffer thoroughly by shaking before use. Buffer WA and buffer WB are supplied as concentrates. Before using for the first time add 20 ml of ethanol (96-100%) to WA and 40 ml of ethanol (96-100%) to WB to obtain a working solution. Extraction recipe 1- Cut 25 mg human or animal tissue (up to 10 mg spleen) into small pieces. Note: For tissues such as spleen with a very high number of cells for a given mass of tissue, no more than 10 mg starting material should be used. For rodent tails, a maximum of 1.2 cm (mouse) or 0.6 cm (rat) tail should be used. When purifying DNA from the tail of an adult mouse or rat, it is recommended to use only cm. 2- Grind the samples using a mortar and pestle to obtain a fine powder under liquid nitrogen or homogenize the samples using a mechanical homogenizer (Polytron, Ultra-Turrax ). a. To grind the samples under liquid nitrogen, Place the sliced sample material into a grinding jar (mortar). Add liquid nitrogen to the mortar and freeze. Grind the sample under liquid nitrogen, and disrupt carefully until it is powdered completely. Decant tissue powder and liquid nitrogen into 1.5 ml microcentrifuge tube. Allow the liquid nitrogen to evaporate, but do not allow the tissue to thaw, and proceed immediately to step 3. Note: Disruption and homogenization time depends on the tissue samples. Disrupt the sample completely until no tissue clumps are visible. Clumps of tissue sample will be difficult to lyse properly and will result in a lower yield of DNA. Do not let the samples thaw during disruption and homogenization step to avoid of DNA degradation. b. To homogenize the sample using a homogenizer: Add 25 mg of tissue to a 1.5 ml microcentrifuge tube (not provided), add 75 μl phosphate buffered saline (PBS) and homogenize. 3- Add 200 μl Buffer TL and 10 μl Proteinase K solution. Vortex to mix. Be sure that the samples are completely covered with lysis solution. Note: in the case of 2b, add 100 μl Buffer TL and 10 μl Proteinase K solution and mix thoroughly by vortexing. 4- Incubate the mixture at 60 ᵒC for 1-3 hours until the tissue is completely lysed. Vortex occasionally during incubation to help the tissue disruption or use a shaking incubator. Note: lysis time varies depending on the type of tissue, but in the most cases Lysis procedure is usually complete in 1 3 h or, for rodent tails, 6 8 h. Samples can be incubated overnight as well. 9

11 After incubation the lysate may appear viscose but it should not be gelatinous to clog the spin column. If the lysate appears very gelatinous, increase the amount of TL buffer and mix well by pipetting until the gelatinous state is eliminated. If more volume of TL buffer is needed, please consider that keeping of the ratio of TL/BL/Ethanol to 1/1/1 is crucial. For RNA rich animal tissues like kidney, liver, spleen, heart and etc, an RNase digestion may be necessary to obtain RNA-free DNA: to perform an RNase treatment: Add 5 μl RNase A (100 mg/ml) solution (not provided) to the lysate and incubate for an additional 5 min at room temperature. 5- Add 200 μl of buffer BL to the lysate, mix well by vortexing (15s) or pipetting (at least 10 times) to obtain a homogenous solution and incubate at 70 ᵒC for 10 min. Note: Do not vortex vigorously to avoid DNA shearing. In order to assure efficient lysis, it is important that the lysate sample and Buffer BL are mixed thoroughly. 6- Add 200 µl absolute ethanol (96-100%) to the lysate and mix thoroughly by pipetting (at least 10 times) or by gently inverting several times or by pulse vortexing for 15s to homogenize solution until 2 formed separate phases ( ethanol and lysate) are disappeared. Do not vortex vigorously. After mixing briefly centrifuge (short spin) the tube to remove the drops from the lid. Note: mixing of the ethanol and the lysate is so important and essential to prepare good binding conditions of the genomic DNA to the silica column. 7- Place the spin column (green ring) into a 2 ml collection tube (provided). Pipette the whole of mixture into the mini spin column placed in a 2 ml collection tube without wetting the rim. Close the cap gently and centrifuge at >8000 x g (>11000 rpm) for 1 min. Discard the flowthrough. Note: the maximum capacity of the spin column to load lysis/binding solution is 800 µl. for larger amounts load the sample into the spin column in two steps sequentially. If the lysate has not completely passed through the column after centrifugation, centrifuge again at higher speed until the spin column is empty. 8- Place the mini spin column in the same collection (reuse). Add 500 µl of WA buffer, wait for few seconds and centrifuge at >8000 x g (>11000 rpm) for 1 min. Discard the flow-through. - Flow-through is containing buffer BL and therefore is not compatible with bleaches. The buffers BL and WA will form very toxic gases in the contact with bleaches. 10

12 9- Place the mini spin column in the same collection tube. Add 500 µl of WB buffer and centrifuge at >8000 x g (>11000 rpm) for 1 min, and discard the flow-through. 10- Place the mini spin column in the same tube. Add 500 µl of WB buffer and centrifuge at >16000 x g (>14000 rpm) for 3 min. Discard the flow-through and collection tube. Note: it s important to dry the silica membrane, since residual ethanol may interfere with subsequent reactions. This centrifuge step will remove all ethanol from the membrane. Following the centrifuge step, remove the column carefully to avoid the contact with the flow-through. If carryover of ethanol occurs, empty the collection tube, place the mini spin column in it, and centrifuge the empty column at >16000 g (>14000 rpm) for 1 min. 11- Place the mini spin column in a clean 1.5 or 2 ml micro centrifuge tube (not-provided), and pipette 2 x 75 µl of EB buffer directly to the center of silica membrane. Incubate at room temperature for 5 min and centrifuge for 1 min at >11000 rpm. Note: for more information about elution procedure please refer to Appendix 2; elution procedures. Page: 16. DNA extraction from bacteria and Archeaes: Before Starting Perform all steps, including centrifugations, at room temperature. For DNA isolation from gram positive bacteria make enzymatic lysis buffer (20 mm Tris Cl, ph mM Na-EDTA, 1.2 % Triton X-100 and immediately before use add Lysozyme to 20 mg/ml. BL buffer may form a precipitate upon storage if necessary warm to 56 C until the precipitate has fully dissolved. Mix BL buffer thoroughly by shaking before use. Buffer WA and buffer WB are supplied as concentrates. Before using for the first time add 20 ml of ethanol (96-100%) to WA and 40 ml of ethanol (96-100%) to WB to obtain a working solution. Extraction recipe Gram positive bacteria 1- Centrifuge 1-2 ml of overnight bacterial culture (OD600= 0.8-1) for 3 min at 5000 rpm to obtain pellet. Discard supernatant. 11

13 2- Resuspend bacterial pellet in 200 µl of enzymatic lysis buffer (lysozyme added). Incubate at 37 C for at least 60 min. 3- Add 10µl of proteinase K and 200 µl BL buffer to the bacterial suspension. Mix immediately and completely by pipetting and incubate at 60 C for 60 min. Important: It s essential that sample and buffer BL are mixed immediately and thoroughly by pipetting to yield a homogeneous solution. Archeaes and Gram Negative bacteria 1- Centrifuge 1-2 ml of overnight bacterial culture (OD600= 0.8-1) for 3 min at 5000 rpm to obtain pellet. Discard supernatant. 2- Add 200 µl of TL Lysis buffer and 10 µl proteinase K to the pellet. Mix immediately and completely by pipetting or vortexing. Note: It s essential that sample and buffer TL are mixed immediately and thoroughly by pipetting to yield a homogeneous solution. 3- Incubate the mixture at 60 ᵒC for min until the pellet is completely lysed. Note: in the most cases Lysis procedure is usually completed in less than 30 min. After incubation the lysate may appear viscose but it should not be gelatinous to clog the spin column. If the lysate appears very gelatinous, increase the amount of TL buffer and mix well by pipetting until the gelatinous state is eliminated. If more volume of TL buffer is needed, please consider that keeping of the ratio of TL/BL/Ethanol to 1/1/1 is crucial. In order to assure efficient lysis, it is important that the lysate sample and Buffer BL are mixed thoroughly. 4- Add 200 µl absolute ethanol (96-100%) to the lysate and mix thoroughly by pipetting (at least 10 times) or by gently inverting several times or by pulse vortexing for 15s to homogenize solution until 2 formed separate phases ( ethanol and lysate) are disappeared. Do not vortex vigorously. After mixing briefly centrifuge (short spin) the tube to remove the drops from the lid. Note: mixing of the ethanol and the lysate is so important and essential to prepare good binding conditions of the genomic DNA to the silica column. 5- Place the spin column (green ring) into a 2 ml collection tube (provided). Pipette the whole of mixture into the mini spin column placed in a 2 ml collection tube without wetting the rim. Close the cap gently and centrifuge at >8000 x g (>11000 rpm) for 1 min. Discard the flowthrough. - Flow-through is containing buffer BL and therefore is not compatible with bleaches. The buffers BL and WA will form very toxic gases in the contact with bleaches. 12

14 Note: the maximum capacity of the spin column to load lysis/binding solution is 800 µl. for larger amounts load the sample into the spin column in two steps sequentially. If the lysate has not completely passed through the column after centrifugation, centrifuge again at higher speed until the spin column is empty. 6- Place the mini spin column in the same collection (reuse). Add 500 µl of WA buffer, wait for few seconds and centrifuge at >8000 x g (>11000 rpm) for 1 min. Discard the flow-through. 7- Place the mini spin column in the same collection tube. Add 500 µl of WB buffer and centrifuge at >8000 x g (>11000 rpm) for 1 min, and discard the flow-through. 8- Place the mini spin column in the same tube. Add 500 µl of WB buffer and centrifuge at >16000 x g (>14000 rpm) for 3 min. Discard the flow-through and collection tube. Note: it s important to dry the silica membrane, since residual ethanol may interfere with subsequent reactions. This centrifuge step will remove all ethanol from the membrane. Following the centrifuge step, remove the column carefully to avoid the contact with the flow-through. If carryover of ethanol occurs, empty the collection tube, place the mini spin column in it, and centrifuge the empty column at >16000 g (>14000 rpm) for 1 min. 9- Place the mini spin column in a clean 1.5 or 2 ml micro centrifuge tube (not-provided), and pipette 2 x 75 µl of EB buffer directly to the center of silica membrane. Incubate at room temperature for 5 min and centrifuge for 1 min at >11000 rpm. Note: for more information about elution procedure please refer to Appendix 2; elution procedures. Page: 16. Typical DNA yield Yields from 0.05 to 0.25 ml of whole blood will range from 1.2 μg up to 15 μg. This is based upon a normal WBC count range of 4-10 million WBC/ml blood and assuming 6 pg DNA per diploid nucleus. 13

15 Table 4: Expected DNA yield from various source samples using Top general genomic DNA mini purification kit. Source Amount DNA (µg) Animal tissue (Mouse) Liver 25 mg µg Kidney 25 mg µg Heart 25 mg 5-15 µg Muscle 25 mg 3-7 µg Blood samples: Human healthy blood 200 µl µg Goat blood 200 µl 3 µg Sheep blood 200 µl 3-6 µg Buffy coat 250 µl µg Animal cells: Vero cells µg HeLa cells µg Lymphocyte µg Bacteria Archeaes 1.5 ml of culture µg Gram negative bacteria 1.5 ml of culture µg Gram positive bacteria 1.5 ml of culture µg DNA Quality The quality of extracted DNA can be measured on the Agarose gel or by spectrophotometer. The 260/280 ratio of the extracted DNA will be between 1.9 ± 0.2. The suitability of DNA for enzymatic reactions can be determined by restriction digestion with EcoRI enzyme or by PCR reaction with random/specific primers. Appendix 1: Collection, storage and preparation of starting material s guidelines: Preparation of blood samples: If possible, collect blood in EDTA to reduce DNA degradation. However, other anticoagulants such ACD (citrate) and CPDA may also be used successfully. Heparin may be used but is not recommended. If 14

16 blood collection tube is not available, anticoagulant EDTA could be added into blood directly and mixed fully, in which every 0.4 ml EDTA solution per 5 ml blood that 0.04 M EDTA solution should be prepared in advance. The amount of DNA isolated from a blood sample is highly dependent on the number of white blood cells present in the sample and the blood storage conditions. A white blood cell count (WBC) in a healthy individual is usually within the range of white blood cells/ml. On average, 3-12 ug of DNA is isolated from 0.2 ml of fresh blood sample. Samples frozen at -80 C typically exhibit a 15% decrease in DNA yield. For best results, the blood should be stored at 4 C for less than 5 days or be frozen at -80 C on the same day. Whole Blood: for short term storage, keep the freshly drawn blood samples at 4 C for less than 5 days. For best results, the samples should be delivered to the laboratory 1-4 days after collection. For long term storage, blood samples should be stored at -80 C soon after collection. Storing of blood samples at -20 C is not recommended for any length of time. Blood storage at this temperature for as little as 3 days results in decreased DNA yield. Storage at this temperature for 2-4 weeks results in half the yield as the same sample stored at -80 C. Buffy coats: Buffy coat is a leukocyte-enriched fraction of whole blood. Preparing a buffy-coat fraction from whole blood is simple and yields approximately 5 10 times more DNA than an equivalent volume of whole blood. Prepare Buffy coat by centrifuging whole blood at 2500 x g for 10 minutes at room temperature. After centrifugation, 3 different fractions are distinguishable: the upper clear layer is plasma; the intermediate layer is Buffy coat, containing concentrated leukocytes; and the bottom layer contains concentrated erythrocytes. Alternatively, the sample tube containing blood may be placed in a vertical position at 4 C overnight to allow cells to settle. Carefully remove the upper plasma layer and discard. Then, carefully collect the Buffy coat with a pipettor. Keep sample on ice until use. For best results, store fresh samples at 4 C for no longer than 5 days. For long-term storage, freeze Buffy coat samples at -70 o C to -80 o C. Before use, thaw samples quickly in a 37 o C water bath and keep samples on ice until use. Other body fluids: Most biological fluids (e.g., plasma, serum, and urine) and stool samples can be stored at 2 8 C for several hours. Freezing at 20 C or 80 C is recommended for long-term storage. Swabs can be stored dry at room temperature. Preparation of cultured cells for DNA extraction: 15

17 Cells grown in suspension; Transfer the culture fluid into 15 ml or 50 ml of centrifuge tube and pellet the culture by centrifugation for 5 min at 3000 rpm. Remove the supernatant completely and store the cell pellet at -20 or -80 C. Cells grown in monolayer; Cells grown in monolayer can be detached from culture flask (or plate) by either Trypsinization or using a cell scraper. To Trypsinize cells: Remove the medium and wash the cells with preheated (at 37 ) PBS. Then aspirate the PBS and add trypsin solution. After cells have become detached from culture flask (or dish), collect and wash the cells with PBS, then resuspend the washed cell pellet in appropriate volume of PBS or fresh media. Using a cell scrape: detach cells from culture flask or dish. Collect and wash the cells with PBS, Determinate the cell number using cell counter (eg. hemocytometer) and transfer the appropriated number of cells (1 ~ 4 x 10 6 cells) to a new 1.5 ml microcentrifuge tube. Centrifuge up to 3 x 10 6 cells for 5 min at 3000 rpm. Resuspend the pellet in appropriate volume of PBS. Continue with extraction protocol. Preparation of animal tissues for DNA extraction As soon as animal tissue is isolated from the whole live organism, the quality of nucleic acid in the tissue sample may begin to degrade if it is not stored under the appropriate conditions. DNA is less susceptible to degradation than RNA, but precautions should be taken to ensure that the sample is stored correctly. The fresh animal tissue can be used directly to isolation of genomic DNA. But if the tissues are not used immediately, those should be stored with liquid nitrogen (below -196 C) or deep freezer (below - 80 C) for long-term. Appendix2: Elution procedures: In addition to the standard method (recovery rate about %), several modifications are possible to increase yield, concentration, and convenience. Use elution buffer preheated to 70 C for one of the following procedures: High yield: Perform two elution steps with the volume indicated in the individual protocol. About % of bound nucleic acid can be eluted. High concentration: Perform one elution step with 60 % of the volume indicated in the individual protocol. Concentration of DNA will be approximately 30 % higher than with standard elution. The yield of eluted nucleic acid will be about 80 %. High yield and high concentration: Apply half the volume of elution buffer as indicated in the individual protocol, incubate for 3 min and centrifuge. Apply a second aliquot of elution buffer, 16

18 incubate and centrifuge again. Thus, about % of bound nucleic acid is eluted in the standard elution volume at a high concentration. Convenient elution: For convenience, elution buffer of ambient temperature may be used. This will result in a somewhat lower yield (approximately 20 %) compared to elution with heated elution buffer. Elution may also be performed with Tris-EDTA-buffer (TE) of ph equal or higher than 8. This will increase DNA stability especially during long term and / or multi use storage at 4 C or ambient temperature by inhibition of omnipresent DNases. However, EDTA interferes, depending on the final concentration, with certain downstream applications. Note: Elution Buffer EB (10 mm Tris/HCl, ph 8.5) provided with the kit does not contain EDTA. For optimal performance of isolated DNA in downstream applications, we recommend eluting with the supplied elution buffer and storage, especially long term, at -20 C. Freeze-thaw cycles will have no effect on most downstream applications. Possible exceptions are detection of trace amounts of DNA or long-range PCR (e.g., > 10 kbp). Multiple freeze-thaw cycles or storing DNA at 4 C or room temperature may influence detection sensitivities or reaction efficiencies due to DNA shearing or adsorption to surfaces. Troubleshooting Gide This part may be useful to solve problems in your DNA extraction procedure. Problem Column is clogged Comments In subsequent preparations, reduce the amount of starting material and/or increase volume of TL Buffer and homogenization time. Increase g-force and centrifugation time Low yield Storage of starting material DNA yield is dependent on the type, size, age, and storage of starting material. If using blood stored for 7 days at +2 to +8 C or for 1 month at 15 to 25 C, the expected yields will be 10 15% lower than those of freshly isolated blood. Stored tissues at inappropriate temperatures, frozen thawed tissues and old samples 17

19 often result in sheared and degraded DNA yields. Too much or too low starting material Insufficient mixing of sample with lysis/ binding buffer Wrong preparations of WA & WB buffers Some problems in elution buffer In future preparations set the amount of starting material according to suggested amount in the kit manual. Adjusting the ratio of lysis buffer/starting material in suggested ratio will be so important for sufficient lysis of material. Ensure to mix starting material thoroughly with TL buffer, BL buffer and ethanol by pulsed vortexing or pipetting before applying the sample to the Mini spin column. Make sure that ethanol has been added to buffer WA & WB correctly before use. If you use water instead of elution buffer, adjust the ph of water to If you use Tris-Cl 10mM or TE buffer, make sure that the ph is not less than 7.5. Low ratio of A 260/ A 280 Too much starting material Insufficient washing Too high ratio of A 260 /A 280 Reduce the amount of starting material as suggested in kit's manual. Be sure that 2X washing with WB buffer is carried out to increase the DNA purity. Perform the optional RNase treatment in the protocol. 18

20 Technical Assistance and Ordering Information Technical support can be obtained on line via our website: or through e.mail to To on line order refer to our website on Or order via fax: +98 (21) Our address: Science & Technology Park, College of Agriculture and Natural Resources, University of Tehran Product Warranty Topaz gene Research guarantees the performance of all products in the manner described in our product literature. The purchaser must determine the suitability of the product for its particular use. Should any product fail to perform satisfactorily due to any reason other than misuse, Topaz Gene Research will replace it free of charge or refund the purchase price. References: 1- Boom R., et al., Rapid and simple method for purification of nucleic acids, J Clin Microbiol.; 28(3): QIAamp DNA Mini and Blood Mini Handbook, 3- Genomic DNA from tissue, User manual, NucleoSpin Tissue. 19

21 Procedure flowchart For tissue samples: add 250µl of buffer TL and 25 µl of proteinase K. Mix well by pipetting or vortexing. Incubate at 56⁰C for 1-3 h. after incubation add 250µl of buffer BL. Mix well and incubate at 70⁰C for 10 min. For blood, body fluids and cells: add 250µl of buffer BL and 25 µl of proteinasee K, mix and incubate for 15 min at 56⁰C. Add 250 µl of absolute ethanol (96-100%) to the lysate and mix thoroughly by pipetting or vortexing. Apply whole of the mixture to the spin column placed into the collection tube. Centrifuge at >6000 x g (>9000 rpm) for 1 min. Discard the flow-through. Pipette 500 µl of WA buffer to the spin column and centrifuge at >6000 x g (>9000 rpm) for 1 min. Discard the flow-through. Pipette 500 µl of WB buffer to the spin column and centrifuge at >6000 x g (>9000 rpm) for 1 min. Discard the flow-through. Pipette 500 µl of WB buffer to the spin column and centrifuge at >16000 x g (>14000 rpm) for 3 min, and discard the flow-through. Place the mini spin column in a clean 1.5 or 2 ml micro centrifuge tube. Pipette 2 x 75 µl of EB buffer directly to the center of silica membrane. Incubate at room temperature for 5 min. Place the mini spin column in a clean 1.5 or 2 ml micro centrifuge tube. Pipette 2 x 75 µl of EB buffer directly to the center of silica membrane. Incubate at room temperature for 5 min. 20

22 Technical Service and Ordering Information For ordering information visit our website at or us at For technical assistance us at If properly stored (RT), all component of the kit are stable for 12 months. Address: Science & Technology Park, : College of Agriculture and Natural Resources, University of Tehran Tel: +98 (26) Fax: +98 (21) : Postal box: : :