illustra tissue & cells genomicprep Midi Flow Kit For the extraction and purification of genomic DNA from animal tissues and cultured mammalian cells

Transcription

1 GE Healthcare illustra tissue & cells genomicprep Midi Flow Kit For the extraction and purification of genomic DNA from animal tissues and cultured mammalian cells Product booklet See back cover for quick reference protocol card Code: (25 purifications)

2 Page finder 1. Legal 4 2. Handling Safety warnings and precautions Storage conditions Expiry 5 3. Components Kit contents Materials to be supplied by user Equipment to be supplied by user 8 4. Description Introduction The basic principle Product specifications Typical output Protocol Preparation of working solutions Gravity protocol for purification of genomic DNA from animal tissue Centrifugation protocol for purification of genomic DNA from animal tissue Gravity protocol for purification of genomic DNA from mammalian cells Centrifugation protocol for purificiation of genomic DNA from mammalian cells Appendices Calculation of RPM from RCF Tissue homogenization considerations Estimation of cell density Estimation of yield and purity Protocol for concentration of purified sample Troubleshooting guide Related products 45 2

3 7. References 47 Quick Reference Protocol Card Tear off sheet containing protocols for the experienced user for isolating:- A. genomic DNA from animal tissue B. genomic DNA from mammalian cell lines Back Cover 3

4 1. Legal Product use restriction The components of the illustra tissue & cells genomicprep Midi Flow Kit have been designed, developed, and sold for research purposes only. They are suitable for in vitro use only. No claim or representation is intended for its use to identify any specific organism or for clinical use (diagnostic, prognostic, therapeutic, or blood banking). It is the responsibility of the user to verify the use of the illustra tissue & cells genomicprep Midi Flow Kit columns for a specific application range as the performance characteristic of this kit has not been verified to a specific organism. GE, imagination at work and GE monogram are trademarks of General Electric Company. illustra, PuReTaq, FideliTaq, MegaBACE and DYEnamic are trademarks of GE Healthcare companies. All third party trademarks are the property of their respective owners General Electric Company - All rights reserved. Previously published Date of revision June All goods and services are sold subject to the terms and conditions of sale of the comapny within GE Healthcare which supplies them. A copy of these terms and conditions is available upon request. Contact your GE Healthcare representative for the most current information (see back cover for contact details). GE Healthcare UK Limited. Amersham Place, Little Chalfont, Buckinghamshire. HP7 9NA UK. 4

5 2. Handling 2.1. Safety warnings and precautions Warning: For research use only. Not recommended or intended for diagnosis of disease in humans or animals. Do not use internally or externally in humans or animals. All chemicals should be considered as potentially hazardous. Only persons trained in laboratory techniques and familiar with the principles of good laboratory practice should handle these products. Suitable protective clothing such as laboratory overalls, safety glasses, and gloves should be worn. Care should be taken to avoid contact with skin or eyes; if contact should occur, wash immediately with water. See Material Safety Data Sheet and or safety statement(s) for specific recommendations. Use of this product with cells, tissue, or tissue products should be considered bio-hazardous. Follow appropriate safety procedures while using this kit and when handling DNA isolated from these sources. Waste effluents from this kit should be decontaminated with bleach or detergent-based method. Decontamination with bleach may be reactive resulting in foam and emission of ammonia gas and should be carried out in an exhaust hood. Consult local safety regulations for safe disposal of all treated waste Storage All kit components should be stored at room temperature (20 25 C) Once reconstituted, store Proteinase K at 4 C Expiry For expiry date please refer to outer packaging label. Reconstituted Proteinase K in DNase-free water is stable for 4 months or to expiry date on outer packaging label, when stored at 4 C. 5

6 3. Components 3.1. Kit contents Identification Pack Size 5 25 Cat. No. purifications purifications Sample Pack Proteinase K, 1 vial 1 vial lyophilized powder (10 mg) (30 mg) (Black colored cap) Lysis buffer type ml 150 ml (Red colored cap) Load buffer type 1 60 ml 275 ml (Blue colored cap) Elution buffer type 2 30 ml 75 ml (Pink colored cap) Elution buffer type 8 20 ml 100 ml (Provided as a 10 Stock solution) (Silver colored cap) Fast-Flow Genomic column (Blue capped column) NAP-25 Desalting 5 25 column (Clear capped column) Column adapters

7 Refer to the Certificate of Analysis for a complete list of kit components. GE Healthcare supplies a wide range of buffer types across the illustra nucleic acid purification and amplification range. The composition of each buffer has been optimized for each application and may vary between kits. Care must be taken to only use the buffers supplied in the particular kit you are using and not use the buffers supplied in other illustra kits e.g. the Lysis buffer type 14 supplied in the illustra tissue & cells genomicprep Midi Flow Kit is not the same as the Lysis buffers type 1 & 4 supplied in the illustra tissue & cells genomicprep Mini Spin Kit. In order to avoid confusion and the accidental switching of buffers between kits, a numbering system has been adopted that relates to the entire range of buffers available in the illustra purification range. For example there are currently 14 Lysis buffer type Wash buffer type 1 6 and Elution buffer type 1 8, respectively. Please ensure you use the correct type of Lysis, Load and Elution buffer for your purification Materials to be supplied by user 15 ml centrifuge tubes 50 ml centrifuge tubes Centrifuge tube holder RNase A, lyophilized powder DNase-free water 1 PBS buffer (Optional when isolating genomic DNA from cells) Steriflip 0.2 µm filter (Millipore, SCGP ) or equivalent. Requires a vacuum source. (Optional) LabMate buffer reservoir (GE Healthcare, product code ) (Optional for final concentration step) Isopropanol 7

8 3.3. Equipment to be supplied by user Fixed angle rotor centrifuge (4 C) Centrifuge equipped with a swing-out rotor and buckets for 50 ml centrifuge tubes Vortex mixer Hand-held motor-driven homogenizer (Kimbel, part no or equivalent) with probe (part no. 0090) Waterbath set to 60 C Waterbath set to 37 C Iced waterbath (4 C) 8

9 4. Description 4.1. Introduction The illustra tissue & cells genomicprep Midi Flow Kit is designed for high yield extraction and purification of genomic DNA from tissue or cultured mammalian cell lines. The entire procedure, including Lysis, Purification and De-salination, can be completed using a gravitybased or a spin-based method, in as little as three hours. Resultant genomic DNA is of high molecular weight, quality and purity, and is therefore compatible with most molecular biology techniques such as PCR amplification, restriction enzyme digestion and DNA sequencing. In general the gravity method takes slightly longer than the spin-based method but with a 2 20% higher recovery of genomic DNA. The illustra tissue & cells genomicprep Midi Flow Kit utilizes a simple and efficient process, employing a simple Lysis procedure (1), Purification using anion-exchange column chromatography with the illustra Fast-Flow Genomic 250 Column and a trouble-free Desalination process using an illustra NAP-25 Desalting Column. The illustra Fast-Flow Genomic 250 column, used for the Purification step, contains an anion-exchange chromatography medium with excellent flow characteristics, exceptional chemical and ph stability, and a high capacity for biomolecules. Genomic DNA, but not potential impurities, binds to the anion exchange medium at high salt concentrations. The DNA is subsequently eluted from the column in a buffer containing higher salt concentrations. The illustra Fast-Flow Genomic 250 columns are disposable, single-use, and robust for both gravity flow or centrifugation protocols (see sections ). They are supplied pre-equilibrated and have excellent flow characteristics, resulting in reduced hands-on and total process time. The buffer used for pre-equilibration has been optimized to 9

10 prevent the majority of impurities from binding to the resin, leading to higher purity and yield of genomic DNA. A Desalination step based on our illustra NAP-25 Columns is provided in the kit to avoid the need for isopropanol precipitation. The columns contain Sephadex G-25 DNA grade media. The Desalination step is rapid and easy, removing > 99% of the salt from the purified genomic DNA solution. This process is reproducible and may be completed in approximately 15 minutes. The kit contains sufficient reagents and columns for 25 ( ) standard midi prep isolations. Typical yield is µg DNA per milligram of tissue. 10

11 4.2. The basic principle Use of the illustra tissue & cells genomicprep Midi Flow Kit involves the following steps (images have been shown for gravity based method for isolation of genomic DNA from tissue only): 1. Tissue or Cell Preparation 2. Lysis Preparation of illustra Fast-Flow Genomic 250 Column 3. Purification 4. Elution Preparation of illustra NAP-25 Desalting Column 5. De-salination Genomic DNA ready for downstream application 11

12 Step Comments Component 1. Tissue or Cell Preparations Tissue is homogenized in 1 PBS buffer. Cells are washed with 1 PBS buffer and resuspended in 1 TE buffer. 2. Lysis Tissue or cells are lysed with Lysis buffer type 14. Samples are incubated with Proteinase K and then RNase A. Tissue samples are diluted with Load buffer type 1. Cell samples are filtered and then diluted with Load buffer type 1. Lysis buffer type 14 Proteinase K Load buffer type 1 3. Purification The lysate is transferred on to the illustra Fast-Flow Genomic 250 Column. During this step genomic DNA binds to the resin. Load buffer type 1 Illustra Fast-Flow Genomic 250 Column After all the solution has passed through the resin, an additional amount of Load buffer type 1 is added to the column to complete the loading and remove all the impurities. 4. Elution Genomic DNA is eluted from the illustra Fast-Flow Genomic 250 Column using high ionic strength buffer. Elution buffer type 2 5. Desalination Salt is removed by use of the illustra NAP-25 Column and purified genomic DNA is eluted using Elution buffer type 8. illustra NAP-25 Desalting Column Elution buffer type 8 12

13 4.3. Product specifications Sample Tissue Cultured cells Sample Input Size < 200 mg of tissue < cultured cells Capacity > 250 µg > 250 µg Typical Yield µg µg (100 mg rat liver) (2.0 x 10 7 MRC5 cells) Purity (A260:A280) > 1.7 > 1.7 Total Time < 3 hours < 4 hours (For full protocol) Product Size > 48 kb > 48 kb Final Elution 3.5 ml 3.5 ml Volume (After De-salination step) 13

14 4.4. Typical Output Figure 1. Pulse field gel characteristics of purified genomic DNA from rat liver using illustra tissue & cells genomicprep Midi Flow Kit and competitor s kit. M1 M2 illustra competitor M1 M2 M1 is Lambda molecular weight marker; M2 is a Low range pulse field gel marker supplied by NEB (product code N0350S). Figure 2. Pulse field gel characteristics of purified genomic DNA from the human lung fibroblast cell line MRC5 using the illustra tissue & cells genomicprep Midi Flow Kit and a competitor s kit. M1 illustra competitor M2 M1 is a Low range pulse field gel marker supplied by NEB (product code N0350S); M2 is a Lambda marker. 14

15 5. Protocols Note: Solutions and Buffers ARE NOT transferable between GE Healthcare kits, e.g., the composition of the Lysis solution type 14 in the tissues & cells genomicprep Midi Flow Kit is not the same as the Lysis solution type 5 in the blood and cells genomicprep Midi Flow Kit. Use of icons The key below describes the purpose of the icons used throughout the protocol booklet. This icon is used to highlight particularly critical steps within the protocol that must be adhered to. If this advice is not followed it will have a detrimental impact on results. This icon is used to highlight technical tips that will enhance the description of the step. These tips may indicate areas of flexibility in the protocol or give a recommendation to obtain optimum performance of the kit. 15

16 5.1. Preparation of working solutions See Sections 3.2. and 3.3. Materials and Equipment to be supplied by user. Proteinase K Prepare a stock solution of Proteinase K by dissolving in DNase-free water to give a final concentration of 20 mg/ml. To the vial containing 30 mg of proteinase K in add 1.5 ml of DNase-free water. Users of the sample pack should add 0.5 ml DNase-free water to the vial containing 10 mg Proteinase K. Once reconstituted Proteinase K should be stored at 4 C. RNase A Prepare a stock solution of RNase A by dissolving in DNase-free water to give a final concentration of 20 mg/ml e.g., to a vial containing 1 mg RNase A add 50 µl of DNase-free water. Elution buffer type 8 Elution buffer type 8 is provided as a 10 stock solution. Prepare a 1:10 dilution of the Elution buffer type 8. You will require a final volume of 28.5 ml per prep. For each purification to be performed, dilute 3 ml of Elution buffer type 8 with 27 ml DNase free water to give a 1 x working solution. Do not make up more buffer than is required. 16

17 5.2. Gravity protocol for the purification of genomic DNA from animal tissue 1. Tissue Preparation Note: For highly fibrous tissue such as kidney and tail, please see section 6.2. for additional considerations. a. Weigh out approximately 100 mg of tissue and place in a 2 ml centrifuge tube. Slice into small pieces. b. Add 1 ml of 1 PBS and vortex. 1 ml PBS c. Spin for 1 minute at 100 g. 1 minute 100 g d. Discard the supernatant and remove any traces of buffer left in the tube using a pipette. e. Add 500 µl of 1 PBS buffer. 500 µl PBS Note: For mg of tissue, add 1 ml of 1 PBS. f. Homogenize the sample using a handheld homogenizer (Refer to Section 6.2. for homogenization protocol considerations). Homogenize tissue sample Note: It is critical to have a completely homogenized sample to obtain good yield of genomic DNA. 17

18 2. Lysis a. Add 500 µl of Lysis buffer type 14 to the homogenized sample and vortex for seconds at the highest possible speed. 500 µl Lysis buffer type 14 Note: For mg tissue add 1 ml Lysis buffer type 14. b. Add 50 µl of Proteinase K solution, vortex briefly and incubate for hours at 60 C. 50 µl Proteinase K, hours 60 C c. Cool the reaction tubes for 3 minutes in an ice bath. Add 20 µl of RNase A solution (20 mg/ml) and incubate for 15 minutes at 37 C. 3 minutes 4 C 20 µl RNase A 15 minutes 37 C d. Transfer crude lysate to 15 ml centrifuge tube. Dilute the crude lysate with 4 ml of water and 5 ml of Load buffer type 1. 4 ml water 5 ml Load buffer type 1 e. Centrifuge for 15 minutes at g at 4 C to pellet particulates. Retain the supernatant. 15 minutes g 4 C 18

19 Preparation of Fast-Flow Genomic 250 Column a. Remove the top cap from the Fast-Flow Genomic 250 column (column with blue cap) and pour off the excess storage liquid. b. Cut off the recessed part of the bottom tip using a guillotine, 4 mm tube-cutters or scissors. c. Insert the column through the hole of a yellow adapter. Place the column with the adapter in a 50 ml centrifuge tube. The adapter will help maintain the column at mid-height in the tube. 3. Purification a. Transfer the clear lysate containing genomic DNA (obtained from Lysis step) to the Fast- Flow Genomic 250 column. b. Allow the solution to flow through the resin completely. c. Add 5 ml of Load buffer type 1 to the column and allow it to completely flow through the resin before proceeding to the Elution step. 5 ml Load 4. Elution a. Transfer the Fast-Flow Genomic 250 column into a fresh 50 ml centrifuge tube b. Add 2.5 ml of Elution buffer type 2 directly onto the center of the column. Retain eluate, containing genomic DNA. 2.5 ml Elution buffer type 2 19

20 Preparation of the NAP-25 Desalting column a Remove the top cap of the NAP-25 column (column with clear cap) and pour off the excess storage liquid. b. Cut off the recessed part of the bottom tip using a guillotine, 4 mm tube-cutters or scissors. c. Insert the column through the hole of a yellow adapter. Place the column with the adapter in a 50 ml centrifuge tube. The adapter will help maintain the column at mid-height in the tube. d. Equilibrate the column with 25 ml of diluted Elution buffer type 8. Note: This can be conveniently accomplished using LabMate PD-10 buffer reservoir. If the buffer reservoir is maintained only for equilibration of the NAP-25 Desalting Column, the LabMate accessory is reusable. Note: It is critical to equilibrate the NAP-25 Desalting Column since UV absorbing stabilizers are used in column packing. 25 ml Elution buffer type 8 5. De-salination a. Transfer the eluate obtained from the Fast- Flow Genomic 250 column to the NAP-25 Desalting column. b. Allow the solution to completely flow into the gel bed. c. Transfer the NAP-25 Desalting column to a new 50 ml centrifuge tube. 20

21 d. Elute the desalted product containing the genomic DNA using 3.5 ml of diluted Elution Buffer type 8. Note: You may use any low salt buffer of choice but you must use the same buffer for both the Column Preparation and Elution steps. Note: It is very important to use stated volumes (25, 2.5 and 3.5 ml) during for column equilibration, sample loading and sample elution. Lower elution volumes will result in lower recovery without influencing the purity of the product. However using larger elution volumes will elute some salt along with genomic DNA. e. The purified genomic DNA is now ready for downstream applications. For short term storage, place genomic DNA at 4 C. Long term storage should be at -20 C. It is recommended that frozen samples be aliquoted to avoid repeated freeze-thaw cycles. 3.5 ml Elution Buffer type 8 21

22 5.3. Centrifugation protocol for the purification of genomic DNA from animal tissue 1. Tissue Preparation Note: For highly fibrous tissue such as kidney and tail, please see section 6.2. for additional considerations. a. Weigh out approximately 100 mg of tissue Slice into small pieces. Transfer to a 2 ml centrifuge tube. b. Add 1 ml of 1 PBS and vortex. 1 ml PBS c. Centrifuge for 1 minute at 100 g. d. Discard the supernatant and remove any traces of buffer left in the tube using a pipette. 1 minute 100 g e. Add 0.5 ml of 1 PBS. 0.5 ml PBS Note: For mg of tissue, add 1 ml of 1 PBS. f. Homogenize the sample using a handheld homogenizer (Refer to Section 6.2. for homogenization protocol considerations). Homogenize tissue sample Note: It is critical to have a completely homogenized sample to obtain good yield of genomic DNA from the purification process. 22

23 2. Lysis a. Add 500 µl of Lysis buffer type 14 to the homogenized sample and vortex for seconds at the highest possible speed. 500 µl Lysis buffer type 14 Note: For mg tissue add 1 ml Lysis buffer type 14. b. Add 50 µl of Proteinase K. solution, vortex briefly and incubate for hours at 60 C. c. Cool the reaction tubes for 3 minutes in an ice bath. Add 20 µl of RNase A solution (20 mg/ml) and incubate for 15 minutes at 37 C. 50 µl Proteinase K, hours at 60 C 3 minutes 4 C 20 µl RNase A 15 minutes 37 C d. Transfer the crude lysate to a 15 ml centrifuge tube. Dilute the crude lysate with 4 ml of water and 5 ml of Load buffer type 1. 4 ml water 5 ml Load buffer type 1 e. Centrifuge for 15 minutes at g at 4 C to pellet particulates. Retain the supernatant. 15 minutes g 4 C 23

24 Preparation of Fast-Flow Genomic 250 Column a. Remove the top cap from the Fast-Flow Genomic 250 column (column with blue cap) and pour off the excess storage liquid. b. Cut off the recessed part of the bottom tip using a guillotine, 4 mm tube-cutters or scissors. c. Insert the column through the hole of a yellow adapter. Place the column with the adapter in a 50 ml centrifuge tube. The adapter will help maintain the column at mid-height in the tube. 3. Purification a. Transfer the clear lysate containing genomic DNA (obtained from Lysis step) to the Fast- Flow Genomic 250 column. b. Place centrifuge tubes containing the columns into an appropriate bucket rotor and spin for 4 6 minutes at 2 g. Note: After centrifugation, if any solution remains on the the top of the resin, continue centrifugation for 2 5 minutes. c. Add 5 ml of Load buffer type minutes 2 g 5 ml Load buffer type 1 d. Spin for 3 minutes at 7 g. If any buffer remains on top of the resin, continue the centrifugation for 2 5 minutes longer. 3 minutes 7 g 24

25 4. Elution a. Transfer the Fast-Flow Genomic 250 column into a fresh 50 ml centrifuge tube b. Add 2.5 ml of Elution buffer type 2 directly onto the center of the column. Retain eluate containing genomic DNA. 2.5 ml Elution buffer type 2 c. Re-load the centrifuge tubes containing the columns back into the bucket rotor and spin at 7 g for 3 minutes. If necessary continue centrifugation for another 2 5 minutes. Retain eluate containing genomic DNA. Preparation of the NAP-25 Desalting column a Remove the top cap of the NAP-25 column (column with clear cap) and pour off the excess storage liquid. b. Cut off the recessed part of the bottom tip using a guillotine, 4 mm tube-cutters or scissors. c. Insert the column through the hole of a yellow adapter. Place the column with the adapter in a 50 ml centrifuge tube. The adapter will help maintain the column at mid-height in the tube. d. Equilibrate the column with 25 ml of diluted Elution buffer type 8. Note: This can be conveniently accomplished using LabMate PD-10 buffer reservoir. If the buffer reservoir is maintained only for equilibration of the NAP-25 Desalting Column, the LabMate accessory is reusable. 3 minutes 7 g 25 ml Elution buffer type 8 25

26 Note: It is critical to equilibrate the NAP-25 Desalting Column since UV absorbing stabilizers are used in column packing. 5. De-salination a. Transfer the eluate obtained from the Fast- Flow Genomic 250 column to the NAP-25 Desalting column. b. Allow the solution to completely flow into the gel bed. c. Transfer the NAP-25 Desalting column to a new centrifuge tube. d. Elute the desalted product containing genomic DNA using 3.5 ml of diluted Elution Buffer type 8. Note: You may use any low salt buffer of choice but you must use the same buffer for both the Column Preparation and Elution steps. Note: It is very important to use stated volumes (25, 2.5 and 3.5 ml) during for column equilibration, sample loading and sample elution. Lower elution volumes will result in lower recovery without influencing the purity of the product. However using larger elution volumes will elute some salt along with genomic DNA. e. The purified genomic DNA is now ready for downstream applications. For short term storage, place genomic DNA at 4 C. Long term storage should be at -20 C. It is recommended that frozen samples be aliquoted to avoid repeated freeze-thaw cycles ml of Elution Buffer type 8

27 5.4. Gravity protocol for purification of genomic DNA from mammalian cell lines Isolation of genomic DNA from mammalian cells 1. Cell Preparation a. Wash cells twice with 5 ml 1 PBS as follows: Re-suspend the cells in 5 ml 1 PBS buffer by pipeting, spin for 10 minutes at g. Carefully pour off the supernatant. Repeat wash once more. Note: Do not use greater than cells per sample. If you wish to prepare genomic DNA from higher cell numbers, split the sample and perform two or more preparations. b. Re-suspend the cell pellet in 1 ml of diluted Elution buffer type 8 by vortexing for seconds at highest speed. Note: Make sure the pellet is completely resuspended. This step is critical for obtaining the maximum possible yield of genomic DNA. < mammalian cells 5 ml 1 PBS 10 minutes g Repeat 1 ml Elution buffer type 8 Vortex 2. Lysis a. Add 4.5 ml of Lysis buffer type 14 and vortex for seconds. 4.5 ml Lysis buffer type 14 Vortex b. Add 50 µl of Proteinase K and vortex briefly (2 seconds). 50 µl Proteinase K Vortex c. Incubate for hours at 60 C hours 60 C 27

28 Note: After the lysis process is complete, a clear solution should be obtained. If the solution is not clear continue the incubation. d. Cool the tube in an ice bath for 2 minutes and add 20 µl of RNase A (20 mg/ml). e. Incubate for 15 minutes at 37 C. 2 minutes 4 C 20 µl RNase A 15 minutes 37 C f. Filter the sample using a Millipore Steriflip filter (0.22 µm) or equivalent before proceeding to purification step. (See section 3.2. for ordering details). Filtration of Crude Lysate using a Steriflip filter g. Connect the Steriflip filter to a vacuum line. h. Wet the filter membrane by adding 1 ml of Load buffer type 1. i. Filter the crude lysate (from step 2.e). j. Wash the membrane using 4 ml of Load Buffer type 1. 1 ml Load buffer type 1 Filter lysate 4 ml Load buffer type 1 K. Disconnect the vacuum line, mix the solution by shaking the tube. Note: Any alternative filtration units with a membrane which has very low DNA binding properties may be used. This filtration step will reduce the possibility of column clogging, particularly when lysis is performed using high densities of cells. If the cell density is low, filtering is optional, although column flow rate may be slowed. 28

29 Preparation of Fast-Flow Genomic 250 Column a. Remove the top cap from the Fast-Flow Genomic 250 column (column with blue cap) and pour off the excess storage liquid. b. Cut off the recessed part of the bottom tip using a guillotine, 4 mm tube-cutters or scissors. c. Insert the column through the hole of a yellow adapter. Place the column with the adapter in a 50 ml centrifuge tube. The adapter will help maintain the column at mid-height in the tube. 3. Purification a. Transfer the clarified lysate containing genomic DNA (obtained from Lysis step) to the Fast-Flow Genomic 250 column. b. Allow the solution to flow through the resin completely. c. Add 5 ml of Load buffer type 1 to the column and allow it to completely flow through the resin before proceeding to the Elution step. 5 ml Load buffer type 1 4. Elution a. Transfer the Fast-Flow Genomic 250 column into a fresh 50 ml centrifuge tube b. Add 2.5 ml of Elution buffer type 2 directly on to 2.5 ml Elution directly onto the center of the column. Retain eluate containing the genomic DNA. 2.5 ml Elution buffer type 2 29

30 Preparation of the NAP-25 Desalting column a Remove the top cap of the NAP-25 column (column with clear cap) and pour off the excess storage liquid. b. Cut off the recessed part of the bottom tip using a guillotine, 4 mm tube-cutters or scissors. c. Insert the column through the hole of a yellow adapter. Place the column with the adapter in a 50 ml centrifuge tube. The adapter will help maintain the column at mid-height in the tube. d. Equilibrate the column with 25 ml of diluted Elution buffer type 8. Note: This can be conveniently accomplished using LabMate PD-10 buffer reservoir. If the buffer reservoir is maintained only for equilibration of the NAP-25 Desalting Column, the LabMate accessory is reusable. Note: It is critical to equilibrate the NAP-25 Desalting Column since UV absorbing stabilizers are used in column packing. 25 ml Elution buffer type 8 5. De-salination a. Transfer the eluate obtained from the Fast- Flow Genomic 250 column to the NAP-25 Desalting column. b. Allow the solution to completely flow into the gel bed. c. Transfer the NAP-25 Desalting column to a new 50 ml centrifuge tube. 30

31 d. Elute the desalted product using 3.5 ml of diluted Elution Buffer type 8. Note: You may use any low salt buffer of choice but you must use the same buffer for both the Column Preparation and Elution steps. Note: It is very important to use stated volumes (25, 2.5 and 3.5 ml) during for column equilibration, sample loading and sample elution. Lower elution volumes will result in lower recovery without influencing the purity of the product. However using larger elution volumes will elute some salt along with genomic DNA. e. The purified genomic DNA is now ready for downstream applications. For short term storage, place genomic DNA at 4 C. Long term storage should be at -20 C. It is recommended that frozen samples be aliquoted to avoid repeated freeze-thaw cycles. 3.5 ml Elution Buffer type 8 31

32 5.5. Centrifugation protocol for purification of genomic DNA from mammalian cell lines Isolation of genomic DNA from mammalian cells 1. Cell preparation a. Wash cells twice with 5 ml 1 PBS as follows: Re-suspend the cells in 5 ml 1 PBS buffer by pipeting, spin for 10 minutes at g. Carefully pour off the supernatant. Repeat wash once more. Note: Do not use greater than cells per sample. If you wish to prepare genomic DNA from higher cell numbers, split the sample and perform two or more preparations. b. Re-suspend the cell pellet in 1 ml of diluted Elution buffer type 8 by vortexing for seconds at highest speed. Note: Make sure the pellet is completely resuspended. This step is critical for obtaining the maximum possible yield of genomic DNA. < mammalian cells 5 ml 1 PBS 10 minutes g Repeat 1 ml Elution buffer type 8 Vortex 2. Lysis a. Add 4.5 ml of Lysis buffer type 14 and vortex for seconds. 4.5 ml Lysis buffer type 14 Vortex b. Add 50 µl of Proteinase K and vortex briefly (2 seconds). 50 µl Proteinase K Vortex hours 60 C c. Incubate for hours at 60 C hours 60 C 32

33 Note: After the lysis process is complete, a clear solution should be obtained. If the solution is not clear continue the incubation. d. Cool the tube in an ice bath for 2 minutes and add 20 µl of RNase A (20 mg/ml). e. Incubate for 15 minutes at 37 C. 2 minutes 4 C 20 µl RNase A 15 minutes 37 C f. Connect the Millipore Steriflip filter to a vacuum g. Wet the filter membrane by adding 1 ml of Load buffer type 1. h. Filter the crude lysate (from step 2.e). i. Wash the membrane using 4 ml of Load Buffer type 1. 1 ml Load buffer type 1 Filter lysate 4 ml Load buffer type 1 j. Disconnect the vacuum line, mix the solution by shaking the tube. Note: Any alternative filtration units with a membrane which has very low DNA binding properties may be used. This filtration step will reduce the possibility of column clogging, particularly when lysis is performed using high densities of cells. If the cell density is low, filtering is optional, although column flow rate may be slowed. 33

34 Preparation of Fast-Flow Genomic 250 Column a. Remove the top cap from the Fast-Flow Genomic 250 column (column with blue cap) and pour off the excess storage liquid. b. Cut off the recessed part of the bottom tip using a guillotine, 4 mm tube-cutters or scissors. c. Insert the column through the hole of a yellow adapter. Place the column with the adapter in a 50 ml centrifuge tube. The adapter will help maintain the column at mid-height in the tube. 3. Purification a. Transfer the clear lysate containing genomic DNA (obtained from Lysis step) to the Fast- Flow Genomic 250 column. b. Place centrifuge tubes containing the columns into an appropriate bucket rotor and spin for 4-6 minutes at 2 g. Note: After centrifugation, if any solution remains on the the top of the resin, continue centrifugation for 2-5 minutes. c. Add 5 ml of Load buffer type 1. d. Spin for 3 minutes at 7 g. If any buffer remains on top of the resin, continue the centrifugation for 2 5 minutes longer. 4. Elution a. Transfer the Fast-Flow Genomic 250 column into a fresh centrifuge tube. 4-6 minutes 2 g 5 ml Load buffer type 1 3 minutes 7 x g 34

35 b. Add 2.5 ml of Elution buffer type 2 directly onto the center of the column. 2.5 ml Elution Buffer type 2 c. Reload the centrifuge tubes containing the columns back into the bucket rotor and spin at for 3 minutes at 7 g. If necessary, continue centrifugation for another 2 5 minutes. Retain eluate containing genomic DNA. Preparation of the NAP-25 Desalting column a Remove the top cap of the NAP-25 column (column with clear cap) and pour off the excess storage liquid. b. Cut off the recessed part of the bottom tip using a guillotine, 4 mm tube-cutters or scissors. c. Insert the column through the hole of a yellow adapter. Place the column with the adapter in a 50 ml centrifuge tube. The adapter will help maintain the column at mid-height in the tube. d. Equilibrate the column with 25 ml of diluted Elution buffer type 8. Note: This can be conveniently accomplished using LabMate PD-10 buffer reservoir. If the buffer reservoir is maintained only for equilibration of the NAP-25 Desalting Column, the LabMate accessory is reusable. Note: It is critical to equilibrate the NAP-25 Desalting Column since UV absorbing stabilizers are used in column packing. 3 minutes 7 g Collect eluate 25 ml Elution buffer type 8 35

36 5. De-salination a. Transfer the 2.5 ml of eluate obtained from the Fast-Flow Genomic 250 column to the NAP-25 Desalting column. b. Allow the solution to completely flow into the gel bed. c. Transfer the NAP-25 Desalting column to a new 50 ml centrifuge tube. d. Elute the desalted product using 3.5 ml of diluted Elution Buffer type 8. Note: You may use any low salt buffer of choice but you must use the same buffer for both the Column Preparation and Elution steps. Note: It is very important to use stated volumes (25, 2.5 and 3.5 ml) during for column equilibration, sample loading and elution. Lower elution volumes will result in lower recovery without influencing the purity of the product. However using larger elution volumes will elute some salt along with genomic DNA. e. The purified genomic DNA is now ready for downstream applications. For short term storage, place genomic DNA at 4 C. Long term storage should be at -20 C. It is recommended that frozen samples be aliquoted to avoid repeated freeze-thaw cycles. 3.5 ml Elution Buffer type 8 36

37 6. Appendices 6.1. RPM calculation from RCF The appropriate centrifugation speed for a specific rotor can be calculated from the following formula: RPM = (RCF/1.12r) Where RCF = relative centrifugal force; r = radius in mm measured from the center of the spindle to the bottom of the rotor bucket; and RPM = revolutions per minute. For example, if an RCF of 735 g is required using a rotor with a radius of 73 mm, the corresponding RPM would be Tissue homogenization considerations Efficient homogenization of tissue samples is important for high DNA yield. Hand-held battery powered homogenizers are relatively inexpensive, and recommended for consistent high yields. Highly fibrous and bony tissues, such as kidney and tails, should be pulverized in liquid nitrogen inside a mortar and pestle that has itself been pre-chilled with liquid nitrogen (2) as outlined below. 1. Weigh an appropriate amount ( mg) of animal tissue in a clean weighing boat. Using a sterile blade, slice the tissue into small pieces while keeping the tissue sample on ice. 2. Transfer the tissue slices to a pestle that has been pre-chilled with liquid nitrogen. Slowly pour liquid nitrogen to cover the entire material. Put the pestle on a bench top and slowly begin crushing the tissue samples to fine powder with the aid of a mortar. It may be necessary to add liquid nitrogen a few times (e.g. if the liquid nitrogen evaporates quickly) to completely pulverize the tissue samples. 37

38 3. After evaporation of remaining liquid nitrogen, transfer the crushed tissue into a 15 ml centrifuge tube add 0.5 ml 1 PBS and continue with homogenization as outlined in the protocols or Tissue Preparation step f. onwards. 38

39 6.3. Estimation of cell density Do not load greater than lysed cells per column when purifying genomic DNA from mammalian cultured cells using the illustra tissue & cells genomicprep Midi Flow Kit. Genomic DNA yields drop when the purification column is overloaded. Absolute numbers of cells that may be lysed per column is cell line dependant. Therefore we recommend that when isolating genomic DNA from a cell line for the first time or for maximal yield, do not exceed cells. If necessary perform 2 parallel purifications. Cell density should be estimated using an automated cell counter (e.g. Coulter) or counted under a microscope using a standard hemocytometer (for example, Hausser Scientific, catalogue number 1483). Follow the guidelines below for measuring cell density using a hemocytometer. 1. Clean a hemocytometer and the short coverslip thoroughly and wipe clean with Ethanol (this step is not necessary if using disposable hemocytometers). 2. If working with adherent cells, treat the cells with trypsin and wash once with PBS. 3. Re-suspend cells in an appropriate volume of PBS to give approximately cells/ml. Make sure the cells are completely re-suspended without any visible clumps. 4. Add 10 µl of re-suspended cells to the hemocytometer, making sure the solution spreads completely under the coverslip (by capillary action). 5. Place the hemocytometer under a light microscope, focus on one grid and the cells using lowest magnification and begin counting cells only at the four corner squares and the middle square (3). Count all cells except those touching the middle lines at the bottom and right. Aim to have cells per grid. If cell count is > 150/ 39

40 grid, it is advisable to dilute the cells, clean the hemocytometer and re-count cells. 6. Add the number of cells in a total of ten grids and multiply by dilution factor supplied with your particular hemocytometer to give the number of cells/ml of PBS Estimation of yield and purity Purified genomic DNA concentration should be determined by UV spectrophotometry (A 260 ) and by agarose gel electrophoresis through comparison with a known standard. The reliable range of A 260 data should be determined for individual spectrophotometers. Generally, for spectrophotometers with a 1 cm path length, A 260 readings should lie between 0.1 and 1.0 and appropriate dilutions (5 to 50 ng/µl) should be analyzed. For Nano-Drop spectrophotometers, absorbance readings between 1 and 10 are reliable. The UV spectrophotometric ratio A 260 /A 280 provides information regarding the purity of genomic DNA. A purity ratio of 1.7 to 1.9 indicates that the genomic DNA is pure for all standard molecular biology applications. If the ratio is lower than 1.7, the purified genomic DNA might contain some protein impurities. Similarly, if the ratio is higher than 1.9, the genomic DNA might contain some RNA impurities. 1 OD unit (A 260 ) is equivalent to approximately 50 µg/ml doublestranded DNA. Yield = A 260 µ 50 µg/ml 0.2 ml = the total µg of purified genomic DNA in the sample. 40

41 6.5. Protocol for concentration of purified sample Should the final sample concentration be too dilute for the selected downstream application, a simple precipitation protocol may be followed: a. Add 1.57 ml (0.7 volumes ) of room temperature Isopropanol to the purified sample. b. Vortex and spin for 15 minutes at g at 4 C. c. Remove the supernatant by decanting, taking care not to disturb the pellet. d. Add 2 ml of 70% ethanol that has been pre-chilled to 4 C. Vortex briefly and spin for 10 minutes at g at 4 C. e. Carefully remove the supernatant without disturbing the pellet. Air dry for 5 10 minutes. Do not over dry the pellet as this will make the DNA difficult to re-dissolve. f. Resuspend the genomic DNA in the desired volume of a suitable buffer (e.g., Tris/EDTA ph 8.0 or 10 mm Tris/HCl ph 8.5). To ensure the pellet is completely dissolved, incubate at 55 C for one hour. 41

42 6.6. Troubleshooting guide This guide may be helpful in the first instance, however if problems persist or for further information please contact GE Healthcare technical services. Telephone numbers are on the back page. Alternatively log onto Problem: DNA yield is low Possible cause Suggestions Homogenization was not complete Tissue lysis and DNA yield is mostly dependent on effective homogenization. Use of a hand-held motor homogenizer is recommended. For difficult to lyse tissues such as mouse or rat tails, crushing the animal tissue in liquid nitrogen is recommended prior to proceeding with DNA extraction (see section 6.2). Use freshly frozen animal tissue for high DNA yield. Use of older samples or tissue that has been repeatedly thawed can reduce DNA yield. Problem: illustra Fast-Flow Genomic 250 column blocked Possible Cause Suggestions Too much sample was processed Pre-filter the sample with a 0.2 m filter prior to loading the column. Do not exceed the input amounts stated in Section

43 Problem: No pellet obtained after centrifugation in the Lysis step Possible cause Suggestions Tissue sample is fully homogenized but now particulates are present If the sample is homogenized a pellet may not be formed. This centrifugation step is done essentially to remove all the insoluble material from the crude lysate which could clog the column. Problem: Reduced illustra Fast-Flow Genomic 250 column flow during Purification step Possible Cause Suggestions Insoluble particles were not completely removed before loading the solution on the column Insoluble particles can slow the flow considerably. If it is too slow, switch to the centrifugation method which will help to improve the flow rate. If necessary use a slightly higher centrifugation speed (i.e., g). N.B. Increasing the centrifugation speed can considerably reduce the yield since the buffers cannot interact with the resin. 43

44 Problem: too much Elution Buffer type 2 and/or 8 was used Possible Cause Suggestions Genomic DNA was eluted with more than 2.5 ml of Elution Buffer type 2 from the fastflow Genomic 250 column. Genomic DNA was eluted with more than 3.5 ml of Elution Buffer type 8 during desalination step This will not adversely affect the purity or the yield of the product. However use only 2.5 ml of the eluate during the De-salination step. If more than 3.5 ml of elution buffer is added to the NAP-25 desalting column, collect only the first 3.5 ml of eluate. If more than 3.5 ml is collected, the final product might contain salt. Problem: Concentration of DNA is too low for a specific application Possible Cause Suggestions Insufficient quantity of starting sample Concentrate sample using protocol in section 6.5. Problem: DNA does not cut to completion with restriction enzymes Possible Cause Suggestions EDTA can inhibit the restriction enzymes Equilibrate and elute the sample from the NAP-25 desalting column with water or Tris buffer instead of Elution buffer type 8. 44

45 6.6. Related products A full range of molecular biology reagents can be found in the GE Healthcare catalog and on the website. If you need further information, GE technical services are happy to assist (world-wide phone numbers can be found on the back cover). Application Product Number Product Pack sizes Kits containing ready-to-use mix for PCR amplification Premixed nucleotides for PCR amplification illustra Hot Start Master Mix illustra PuReTaq Ready-To-Go PCR Beads illustra PuReTaq Ready-To-Go PCR Beads E71182 E71183 FideliTaq PCR Master Mix (2 ) FideliTaq PCR Master Mix Plus illustra DNA Polymerization Mix 100 reactions 96 reactions in 0.2 ml tubes/plate 5 96 reactions in 0.2 ml tubes/plate 100 reactions 100 reactions 10 µmol 45

46 Application Product Number Product Pack sizes Premixed nucleotides for PCR amplification Preparation of PCR products for automated sequencing Sequencing reaction kits optimized for MegaBACE DNA analysis system illustra DNA Polymerization Mix illustra PCR Nucleotide Mix illustra PCR Nucleotide Mix 40 µmol (4 10 µmol) 500 µl 1 ml US78200 ExoSAP-IT TM 100 reactions US78201 ExoSAP-IT 500 reactions US81050 US81060 DYEnamic ET Terminator Cycle Sequencing Kits DYEnamic ET Terminator Cycle Sequencing Kits 100 templates templates 46

47 7. References 1. Alijanabi, S.M. and Martinez, I., Nucl, Acids Res. 25, (1997). 2. Sambrook, J and Russell, D.W., Molecular Cloning, A Laboratory Manual, chapter 6, (2001). 47

48 GE Healthcare offices: GE Healthcare Bio-Sciences AB Björkgatan Uppsala Sweden GE Healthcare Europe GmbH Munzinger Strasse 5 D Freiburg Germany GE Healthcare UK Limited Amersham Place Little Chalfont Buckinghamshire HP7 9NA UK GE Healthcare Bio-Sciences Corp 800 Centennial Avenue P.O. Box 1327 Piscataway NJ USA GE Healthcare Bio-Sciences KK Sanken Bldg Hyakunincho Shinjuku-ku Tokyo Japan GE Healthcare regional office contact numbers: Asia Pacific Tel: Fax: Australasia Tel: Fax: Austria Tel: 01/ Fax: 01/ Belgium Tel: Fax: Canada Tel: Fax: Central & East Europe Tel: Fax: Denmark Tel: Fax: Eire Tel: Fax: Finland & Baltics Tel: Fax: France Tel: Fax: Germany Tel: Fax: Greater China Tel: Fax: Italy Tel: Fax: Japan Tel: Fax: Korea Tel: Fax: Latin America Tel: Fax: Middle East & Africa Tel: Fax: Netherlands Tel: Fax: Norway Tel: Fax: Portugal Tel: Fax: Russia, CIS & NIS Tel: Fax: Spain Tel: Fax: Sweden Tel: Fax: Switzerland Tel: Fax: UK Tel: Fax: USA Tel: Fax: GE Healthcare UK Limited Amersham Place, Little Chalfont, Buckinghamshire, HP7 9NA, UK imagination at work PL Rev E 2007

49 The next four pages are a protocol card. Please add to the back page as a tear off addition.

50 Quick Reference Protocol Card illustra tissue & cells genomicprep Mini Spin Kit A. Gravity protocol for the purification of genomic DNA from animal tissue Ensure 20 mg/ml Proteinase K and RNase A available Ensure appropriate volume of diluted Elution buffer type 8 available :Homogenize :Add :Spin :Incubate (25 purifications) 1. Tissue Preparation 100 mg tissue into 2 ml centrifuge tube 1 ml PBS; vortex 1 minute 100 g; discard supernatant, removing any trace of buffer 0.5 ml PBS Homogenize 2. Lysis 500 µl Lysis buffer type 14; vortex 50 µl Proteinase K; vortex hours at 60 C Cool 3 minutes in ice bath 20 µl RNase A 15 minutes 37 C 4 ml water & 5 ml Load buffer type 1 15 minutes g 4 C; retain supernatant Preparation of Fast-Flow Genomic 250 Column

51 Remove top cap and pour off excess storage liquid Cut off bottom tip and load column into 50 ml centrifuge tube 3. Purification Load clarified lysate onto column; allow solution to flowthrough completely 5 ml Load buffer type 1; allow solution to flowthrough completely 4. Elution Transfer column to a fresh 50 ml centrifuge tube 2.5 ml Elution buffer type 2; retain eluate Preparation of NAP-25 De-salting Column Remove top cap and pour off excess storage liquid Cut off bottom tip and load column into 50 ml centrifuge tube 25 ml Elution buffer type 8 5. De-salination Load eluate from step 4 onto column; allow to flow into resin Transfer column to a fresh 50 ml centrifuge tube 3.5 ml Elution buffer type 8 Store purified genomic DNA at 4 C short term or -20 C long term imagination at work PC Rev-E 2007

52 Remove top cap and pour off excess storage buffer Cut off bottom tip and load column into 50 ml centrifuge tube 3. Purification Load clarified lysate onto column; allow solution to flowthrough completely 5 ml Load buffer type 1; allow solution to flowthrough completely 4. Elution Transfer column to a fresh 50 ml centrifuge tube 2.5 ml Elution buffer type 2; retain eluate Preparation of NAP-25 De-salting Column Remove top cap and pour off excess storage buffer Cut off bottom tip and load column into 50 ml centrifuge tube 25 ml Elution buffer type 8 5. De-salination Load eluate from step 4 onto column; allow to flow into gel bed Transfer column to a fresh 50 ml centrifuge tube 3.5 ml Elution buffer type 8 Store purified genomic DNA at 4 C short term or -20 C long term GE, imagination at work and GE monogram are trademarks of General Electric Company. illustra is a trademark of GE Healthcare Companies General Electric Company-All rights reserved. First published 2006 GE Healthcare reserves the right, subject to any regulatory and contractual approval, if required, to make changes in specifications and features shown herein, or discontinue the product described at any time without notice or obligation. Contact your GE Healthcare representative for the most current information and a copy of the terms and conditions. GE Healthcare UK Limited. Amersham Place, Little Chalfont, Buckinghamshire, HP7 9NA UK

53 Quick Reference Protocol Card illustra tissue & cells genomicprep Mini Spin Kit B. Gravity protocol for the purification of genomic DNA from mammalian cells Ensure 20 mg/ml Proteinase K and RNase A available Ensure appropriate volume of diluted Elution buffer type 8 available :Homogenize :Add :Spin :Incubate 1. Cell preparation Count cells 5 ml PBS; re-suspend by pipeting 10 minute g; discard supernatant 5 ml PBS; re-suspend by pipeting 10 minute g; discard supernatant 1 ml Elution buffer type 8; vortex seconds 2. Lysis (25 purifications) 4.5 ml Lysis buffer type 14; vortex 50 µl Proteinase K; vortex hours at 60 C Cool 3 minutes in ice bath 20 µl RNase A 15 minutes 37 C Filter using a Milipore steriflip filter (pre-wet filter with 1 ml Load buffer type 1, add crude lysate & wash with 4 ml Load buffer type 1) Preparation of Fast-Flow Genomic 250 Column