Protocol (50 prep) - Norgen Biotek Corp.
Contents
1. Parkway Thorold ON Canada L2V 4Y6 Phone 905 227 8848 Fax 905 227 1061 Toll Free in North America 1 866 667 4362 2014 Norgen Biotek Corp P127300 9 M142. viable yeast bacterial cells or 50 mg wet weight of fungal culture Ensure that the dry spin under the Column Wash procedure Ethanol is performed in order to remove traces of ethanol prior to carryover elution Ethanol is known to interfere with many downstream applications DNA does not perform well in The column was Traces of salt from the binding step may remain in the downstream not washed twice sample if the column is not washed twice with the Wash applications with the provided Solution A Salt may interfere with downstream applications and thus must be washed from the column PCR reaction conditions need to be optimized Take steps to optimize the PCR conditions being used including varying the amount of template 20 ng to 50 ng for 20 uL of PCR reaction changing the source of Taq polymerase adding BSA final concentration is 0 1 yg ul looking into the primer design and adjusting the annealing conditions Related Products Product HighRanger 1kb DNA Ladder 11900 UltraRanger 1kb DNA Ladder 12100 Bacterial Genomic DNA Isolation Kit 17900 Plant Fungi DNA Isolation Kit 26200 Technical Support Contact our Technical Support Team between the hours of 8 30 and 5 30 Eastern Standard Time at 905 227 8848 or Toll Free at 1 866 667 4362 Technical support can also be obtained from our website www norgenbiotek com or through email at techsupport norgenbiotek com 3430 Schmon
3. we recommend using 20 50 ng of DNA and adding BSA to the PCR mix to a final concentration of 0 1 ug uL 5 Storage of DNA The purified nucleic acids may be stored at 20 C for a few days It is recommended that samples be placed at 70 C for long term storage Troubleshooting Guide Problem Possible Cause Solution and Explanation Too many cells were applied to the column Ensure that The micro the amount of cells used is less than 1 x 10 viable yeast spin column The sample is cells or 50 mg wet weight of fungal culture Clogging can is clogged too large be alleviated by increasing the g force and or centrifuging for a longer period of time until the lysate passes through the column Turbid elution The sample is too large Depending on fungi species sometimes turbidity can be observed in the elution This may inhibit downstream application Reduce the amount of cells used and perform a third wash during the Column Wash step Lysis was not completed Increase the incubation time at 65 C to 15 minutes Ethanol was not Ensure that 42 mL of 96 100 ethanol is added to the Wash Solution A DARON SAE A A supplied Wash Solution A prior to use Solution Bx was Solution BX enhances DNA binding to the column for not added to the isate maximum DNA recovery ysa The sample is Too many cells were applied to the column Ensure that too large the amount of cells used is less than 1 x 10
4. e bacteria e No phenol or chloroform extractions e Yields high quality DNA that is ready for PCR and other downstream applications Kit Components Component Product 27300 50 preps Lysis Buffer L 30 mL Resuspension Solution A 20 mL Solution BX 28 mL Wash Solution A 18 mL Elution Buffer B 15 mL Bead Tubes 50 Spin Columns 50 Collection Tubes 50 Elution tubes 1 7 mL 50 Product Insert 1 Storage Conditions and Product Stability All solutions should be kept tightly sealed and stored at room temperature These reagents should remain stable for at least 1 year in their unopened containers Precautions and Disclaimers This kit is designed for research purposes only It is not intended for human or diagnostic use Ensure that a suitable lab coat disposable gloves and protective goggles are worn when working with chemicals For more information please consult the appropriate Material Safety Data Sheets MSDSs These are available as convenient PDF files online at www norgenbiotek com Customer Supplied Reagents and Equipment Benchtop microcentrifuge 1 5 mL microcentrifuge tubes 65 C water bath or heating block 96 100 ethanol Homogenizer or motor and pestle RNase A optional Lyticase optional Procedure All centrifugation steps are carried out in a benchtop microcentrifuge Various speeds are required for different steps so please check your microcentrifuge specificatio
5. e to a provided Bead Tube and secure the tube horizontally on a flat bed vortex pad with tape or in any commercially available bead beater equipment e g MP Biomedicals FastPrep 24 or Scientific Industries Disruptor Genie Vortex for 5 minutes at maximum speed or optimize the condition for any commercially available bead beater equipment Note Foaming during the homogenization is common This foaming is due to detergents present in the Lysis Buffer L and will not affect the protocol Incubate the Bead Tube with lysate at 65 C for 10 minutes Occasionally mix the lysate 2 or 3 times during incubation by inverting the tube Briefly spin the tube to remove liquid from the cap and transfer all of the lysate including cell debris to a DNase free microcentrifuge tube provided by the user by pipetting Ensure that the beads are not transferred during the pipetting Centrifuge the tube for 2 minute at 14000 x g 14 000 RPM Carefully transfer clean supernatant to a new DNase free microcentrifuge tube provided by the user without disturbing the pellet Note the volume Add an equal volume of 96 100 ethanol provided by the user to the lysate collected above 100 uL of ethanol is added to every 100 uL of lysate Vortex to mix Add 300 uL of Solution BX and briefly vortex to mix Proceed to Step 2 Binding to Column 1B Lysate Preparation with Lyticase Optional In general no extra enzymes are required to lyse fungal ce
6. g gt lt NORGEN BIOTEK wie CORPORATION Fungi Yeast Genomic DNA Isolation Kit 3430 Schmon Parkway Thorold ON Canada L2V 4Y6 Phone 866 667 4362 e 905 227 8848 Fax 905 227 1061 Email techsupport norgenbiotek com Product Insert Product 27300 Norgen s Fungi Yeast Genomic DNA Isolation Kit is designed for the rapid preparation of genomic DNA from viable yeast cells fungal spores or mycelium and Gram positive bacteria Genomic DNA is efficiently extracted from the cells by a combination of heat treatment detergents and the use of provided Bead Tubes The purified genomic DNA is fully digestible with all restriction enzymes tested and is completely compatible with downstream applications such as PCR and Southern Blot analysis Typical yields of genomic DNA will vary depending on the cell density of the yeast or fungal culture and species The option of an additional lyticase treatment is also provided in order to allow for improved DNA yields for certain fungal and yeast species Preparation time for a single sample is less then 30 minutes and each kit contains sufficient materials for 50 preparations Norgen s Purification Technology Purification is based on spin column chromatography The process involves first adding Lysis Buffer L to the fungi or yeast sample At this point an optional RNase treatment can be performed if RNA free genomic DNA is required Next the mixture is placed into a provided Bead Tube and t
7. he tube is vortexed for 5 minutes in order to efficiently and rapidly homogenize the sample and extract the DNA The sample is then centrifuged and the supernatant is transferred to a DNAse free microcentrifuge tube Ethanol and Solution BX are added to the lysate and it is then loaded onto one of the provided spin columns Norgen s columns bind nucleic acids in a manner that depends on ionic concentrations thus only the DNA will bind to the column while the proteins are removed in the flowthrough or retained on top of the resin The bound DNA is then washed twice using the provided Wash Solution A and the purified DNA is eluted using the Elution Buffer The purified DNA can be used in sensitive downstream applications including PCR and Southern blotting Kit Specification Kit Specifications Maximum Column Binding Capacity 50 ug Maximum Column Loading Volume 650 uL Maximum Amount of Starting Material Fungi wet weight 50 mg Yeast or bacterial culture 0 5mL 1mL Average Yields Pichia sp Yeast 25 ug Botrytis cinerea 32 ug Fusarium sp 42 ug Aspergillus niger 26 ug Mucor racemosus 15 ug Cladosporium cladosporioides 12 ug Penicillium sp 40 ug Rhizopus oryzae 7 ug Alternaria tenuissima 30 ug Time to Complete 10 Purifications 30 minutes Yield will vary depending on the type of sample processed Advantages e Rapid and convenient method to isolate genomic DNA from different fungi yeast and Gram positiv
8. lls when using this kit However the combination of using the Bead Tubes with Lyticase will improve DNA yield for certain fungal and Gram positive bacterial species If low DNA yield is expected please follow the alternative lysis step provided below a b C Follow step 1Aa and 1Ab as above Add 250 uL of Resuspension Solution A to the cell pellet Resuspend the cells by gentle vortexing Add 200 units of Lyticase see Notes prior to use and mix well Incubate at 37 C for 45 minutes Note The time for incubation may vary from 30 minutes to 1 hour Please refer to the Lyticase manufacturer s instruction Proceed to step 1Ac 1C Lysate Preparation Yeast and Gram positive Bacteria a Transfer up to 1 mL of yeast or Gram positive bacterial culture maximum input 1 x 10 cfu s to a microcentrifuge tube and centrifuge at 14 000 x g 14 000 RPM for 1 minute to pellet the cells Pour off the supernatant carefully so as not to disturb or dislodge the cell pellet Proceed to Step 1Ac 2 Binding Nucleic Acids to Column a Obtain a spin column assembled with its collection tube b Apply up to 650 uL of the lysate with ethanol onto the column and centrifuge for 1 minute at 10 000 x g 10 000 RPM Discard the flowthrough and reassemble the spin column with the collection tube Note Ensure the entire lysate volume has passed through into the collection tube by inspecting the column If the entire lysate volume has
9. not passed spin for an additional minute c Repeat step 2b with remained lysate 3 Column Wash a Apply 500 uL of Wash Solution A to the column and centrifuge for 1 minute at 10 000 x g 10 000 RPM Note Ensure the entire wash solution A has passed through into the collection tube by inspecting the column If the entire wash volume has not passed spin for an additional minute Discard the flowthrough and reassemble the column with its collection tube Repeat step 3a to wash column a second time Discard the flowthrough and reassemble the spin column with its collection tube Spin the column for 2 minutes at 14 000 x g 14 000 RPM in order to thoroughly dry the resin Discard the collection tube 2205 4 Nucleic Acid Elution Place the column into a fresh 1 7 mL Elution tube provided with the kit Add 100 uL of Elution Buffer B to the column c Centrifuge for 2 minutes at 10 000 x g 10 000 RPM Note the volume eluted from the column If the entire volume has not been eluted spin the column at 14 000 x g 14 000 RPM for 1 additional minute d Optional An additional elution may be performed if desired by repeating steps 4b and 4c using 100 uL of Elution Buffer B To prevent dilution of the elution it is recommended that a separate elution tube is used for the second elution The total yield can be improved by an additional 20 30 when this second elution is performed o Note For maximum PCR robustness
10. ns to ensure that it is capable of the proper speeds All centrifugation steps are performed at room temperature The correct rom can be calculated using the formula RPM RCF 1 118 x 10 r where RCF required gravitational acceleration relative centrifugal force in units of g r radius of the rotor in cm and RPM the number of revolutions per minute required to achieve the necessary g force Notes prior to use e A variable speed centrifuge should be used for maximum kit performance If a variable speed centrifuge is not available a fixed speed centrifuge can be used however reduced yields may be observed e Ensure that all solutions are at room temperature prior to use and that no precipitates have formed If necessary warm the solutions and mix well until the solutions become clear again e Prepare a working concentration of the Wash Solution A by adding 42 mL of 96 100 ethanol provided by the user to the supplied bottle containing the concentrated Wash Solution A This will give a final volume of 60 mL The label on the bottle has a box that may be checked to indicate that the ethanol has been added e The maximum yeast or bacteria input amount should not exceed 1 x 10 cfu s Depending on culture growth this is equivalent to approximately 0 5 to 1 0 mL of an overnight culture e For the isolation of genomic DNA from fungal species other than yeast Collection Solution must be prepared Collection Solution consi
11. sts of 0 9 w v NaCl prepared with distilled water e Preheat a water bath or heating block to 65 C e Optional The DNA yield from some fungal species may be increased by performing an optional Lyticase lysis step If desired prepare a Lyticase stock solution according to the supplier s instruction It is recommended that the stock solution have a minimum concentration of 4 units per uL Aliquot and store the unused portions at 20 C until needed 1A Lysate Preparation Fungi Growing on Plates or Culture a Fungi Growing on Plates Add approximately 5 mL volume can be adjusted based on density of fungal growth of Collection Solution see notes before use to the plate and gently collect fungal spores and mycelium with an inoculation loop or autoclaved pipette tip ensuring not to collect any agar debris Transfer up to 1 mL of washed spores and wet mycelium to a microcentrifuge tube provided by user Fungi in Culture For fungi growing in a culture transfer 50 mg of fungi wet weight to a microcentrifuge tube b Centrifuge at 14 000 x g 14 000 RPM for 1 minute to pellet the cells Pour off the supernatant carefully so as not to disturb or dislodge the cell pellet Add 500 uL of Lysis Buffer L to the cell pellet Resuspend the cells by gentle vortexing Optional RNase A treatment If RNA free genomic DNA is required add the equivalent of 10 KUnitz of RNase A not to exceed 20 uL to the cell suspension Transfer the mixtur
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