51400 - Protocol (20 prep)
Contents
1. gt k 3430 Schmon Parkway N R F N Thorold ON Canada L2V 4Y6 x Phone 866 667 4362 e 905 227 8848 Fax 905 227 1061 BIOTEK si CORPORATION Email techsupport norgenbiotek com Blood DNA Isolation Midi Kit Product Insert Product 51400 Norgen s Blood DNA Isolation Midi Kit is designed for the rapid preparation of genomic DNA from 0 3 to 2 mL of whole blood Purification is based on spin column chromatography as the separation matrix Norgen s column binds DNA under optimized salt concentrations and releases the bound DNA under low salt and slightly alkali conditions The purified genomic DNA is fully digestible with all restriction enzymes tested and is completely compatible with downstream applications including real time PCR and southern blot analysis Norgen s Blood DNA Isolation Kit allows for the isolation of genomic DNA from the blood of various species including humans The genomic DNA is preferentially purified from other cellular proteinaceous components Typical yields of genomic DNA will vary depending on the cell density of the blood sample Preparation time for a single sample is less than 45 minutes and each kit contains sufficient materials for 20 preparations Kit Components Component Product 51400 20 samples Lysis Buffer B 2x 40 mL Solution WN 55 mL Wash Solution A 2x 38 mL Elution Buffer B 30 mL Proteinase K 4 4mL Midi Spin Columns 20 Midi Collection Tubes 20 Mi2. prior to the next step f Add 1 25 mL of 96 100 Ethanol to the sample and mix vigorously by shaking the tube for 10 seconds then vortex for 10 seconds g Briefly spin the tube to collect any drops of liquid from the inside of the lid 2 Sample Binding to Column Assemble a column with one of the provided collection tubes Apply the lysate to the column and centrifuge for 3 minutes at 1 850 x g 3 000 RPM c Discard the flowthrough Reassemble the column and the collection tube of Note Ensure that all of the lysate has passed through into the collection tube If the entire lysate volume has not passed centrifuge for an additional 2 minutes at 4 500 x g 5 000 RPM o Column Wash a Apply 4 5 mL of Solution WN ensure ethanol was added to the column and centrifuge for 2 minutes at 1 850 x g 3 000 RPM Discard the flowthrough and reassemble the spin column with its collection tube Note Ensure the entire wash solution has passed through into the collection tube by inspecting the column If the entire wash volume has not passed spin for an additional minute at 4 500 x g 5 000 RPM b Apply 4 5 mL of Wash Solution A ensure ethanol was added to the column and centrifuge for 1 minute at 4 500 x g 5 000 RPM Discard the flowthrough and reassemble the spin column with its collection tube Note Ensure the entire wash solution has passed through into the collection tube by inspecting the column If the entire wash
3. volume has not passed spin for an additional minute at 4 500 x g 5 000 RPM c Wash column another time by adding 4 5 mL of Wash Solution A and centrifuging for 1 minute at 4 500 x g 5 000 RPM Discard the flowthrough and reassemble the spin column with its collection tube Note Ensure the entire wash solution has passed through into the collection tube by inspecting the column If the entire wash volume has not passed spin for an additional minute at 4 500 x g 5 000 RPM d Spin the column for 15 minutes in order to thoroughly dry the column at 4 500 x g 5 000 RPM Discard the collection tube DNA Elution Place the column into a provided 15 mL elution tube Add 0 5 mL of Elution Buffer B pre heated at 70 C to the column Centrifuge for 3 minutes at 4 500 x g 5 000 RPM Optional An additional elution may be performed if desired by repeating steps 4a 4d Collect second elution into a new collection tube The yield can be improved by an additional 20 30 when this second elution is performed oops 5 Storage of DNA The purified DNA sample may be stored at 4 C for a few days It is recommended that samples be placed at 20 C for long term storage B Isolation of DNA from 1 2 mL of Blood 1 Sample Preparation NOTE For DNA isolation from blood containing Gram positive bacterial pathogens please see Appendix B for Sample Preparation a Add 0 2 mL of Proteinase K vortex the Proteinase K before us
4. cell lysis Ensure that correct volume of Lysis Buffer B was added to blood sample Also increase incubation time up to 20 25 minutes at 56 C Low DNA binding Ensure ethanol is added to the sample DNA does not perform well in downstream applications DNA was not washed three times with the provided solutions Ensure the column was washed one time with Solution WN and two times with Wash Solution A Ethanol carryover Ensure that the dry spin under the Column Wash procedure is performed in order to remove traces of ethanol prior to elution Ethanol is known to interfere with many downstream applications High DNA input used in PCR reaction For best results make sure that the final concentration of DNA in the PCR reaction does not exceed 75 ng uL 1 5 ug DNA per 20 uL PCR reaction 3430 Schmon 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 P151400 6 M14
5. d to the column and centrifuge for 2 minutes at 1 850 x g 3 000 RPM Discard the flowthrough and reassemble the spin column with its collection tube Note Ensure the entire wash solution has passed through into the collection tube by inspecting the column If the entire wash volume has not passed spin for an additional minute at 4 500 x g 5 000 RPM Apply 4 5 mL of Wash Solution A ensure ethanol was added to the column and centrifuge for 1 minute at 4 500 x g 5 000 RPM Discard the flowthrough and reassemble the spin column with its collection tube Note Ensure the entire wash solution has passed through into the collection tube by inspecting the column If the entire wash volume has not passed spin for an additional minute at 4 500 x g 5 000 RPM Wash column another time by adding 4 5 mL of Wash Solution A and centrifuging for 1 minute at 4 500 x g 5 000 RPM Discard the flowthrough and reassemble the spin column with its collection tube Note Ensure the entire wash solution has passed through into the collection tube by inspecting the column If the entire wash volume has not passed spin for additional minute at 4 500 x g 5 000 RPM Spin the column for 15 minutes in order to thoroughly dry the column at 4 500 x g 5 000 RPM Discard the collection tube DNA Elution Place the column into a provided 15 mL elution tube Add 0 5 mL of Elution Buffer B pre heated at 70 C to the column Centrifuge for 3 mi
6. d should be handled with care Guanidinium salts form highly reactive compounds when combined with bleach thus care must be taken to properly dispose of any of these solutions Blood of all human and animal subjects is considered potentially infectious All necessary precautions recommended by the appropriate authorities in the country of use should be taken when working with blood Customer Supplied Reagents and Equipment e Variable speed swing bucket centrifuge that can reach 4500 x g 5000 rpm and can accommodate 15 mL centrifuge tubes Micropipettors 96 100 ethanol 56 C waterbath or incubator 70 C incubator Lysozyme for blood containing Gram positive bacterial pathogens 37 C incubator for blood containing Gram positive bacterial pathogens Vortex Procedure All centrifugation steps are carried out in a swing bucket centrifuge that can reach 4500 x g 5000 rpm and can accommodate 15 mL centrifuge tubes Various speeds are required for different steps so please check your centrifuge specifications 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 5 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 Flow Chart Procedure for Pur
7. di Elution Tubes 20 Product Insert 1 Specifications Kit Specifications Blood Input 0 3 2 mL Column Binding Capacity gt 100 ug Average Yield 60 ug Time to Complete 10 Purifications 50 60 minutes Yield will vary depending on the type of blood processed Advantages e Fast and easy processing using a rapid spin column format e Isolate high quality genomic DNA free from RNA contamination e Recovered genomic DNA is compatible with various downstream applications 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 The kit contains a ready to use Proteinase K solution which is dissolved in a specially prepared storage buffer The Proteinase K is stable for up to 1 year after delivery when stored at room temperature To prolong the lifetime of Proteinase K storage at 2 8 C is recommended 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 The Lysis Buffer B and Solution WN contain guanidinium salts an
8. e to a 15 mL tube b Transfer 1 2 mL of blood sample to the tube containing Proteinase K c Add 3 mL of Lysis Buffer B to the blood and mix vigorously by shaking the tube for 10 seconds then vortex vigorously for 10 seconds Note During vortex ensure that the contents are being mixed well d Briefly spin the tube to collect any drops of liquid from the inside of the lid e Incubate at 56 C for 20 minutes mixing once during incubation by vigorous shaking of o oops Note If any debris is present in the sample centrifuge for 2 minutes at 4 500 x g 5 000 RPM to precipitate Transfer the clean supernatant to a clean tube prior to the next step Add 2 5 mL of 96 100 Ethanol to the sample and mix vigorously by shaking the tube for 10 seconds then vortex for 10 seconds Briefly spin the tube to collect any drops of liquid from the inside of the lid Sample Binding to Column Assemble a column with one of the provided collection tubes Apply half of the lysate to the column and centrifuge for 3 minutes at 1 850 x g 3 000 RPM Discard the flowthrough Reassemble the column and the collection tube Note Ensure that all of the lysate has passed through into the collection tube If the entire lysate volume has not passed centrifuge for an additional 2 minutes at 4 500 x g 5 000 RPM Repeat Steps 2b and 2c to bind the remainder of the lysate Column Wash Apply 4 5 mL of Solution WN ensure ethanol was adde
9. ifying Blood DNA using Norgen s Blood DNA Isolation Midi Kit Obtain anticoagulated blood sample and transfer into a tube containing Proteinase K Add Lysis Buffer B Vortex Spin briefly Incubate optional SPIN Add Ethanol Bind to column SPIN Wash once with Solution WN Wash twice with Wash Solution A Dry spin SPIN Elute DNA with Elution Buffer B SPIN AA lt 4 T co a et oo e Pure Genomic DNA Notes prior to use 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 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 For best results the use of whole blood collected into tubes containing an anticoagulant is highly recommended e Both fresh and frozen anticoagulated blood may be used with this procedure Ensure that frozen blood is thawed at room temperature prior to starting the protocol e Prepare a working concentration of the Solution WN by adding 73 mL of 96 100 ethanol provided by the user to the supplied bottle containing the concentrated Solution WN This will give a final volume of 128 mL The label on the bottle has a box that may be checked to indicate that the ethanol has been added e Prepare a working c
10. nutes at 4 500 x g 5 000 RPM Optional An additional elution may be performed if desired by repeating steps 4a 4d Collect second elution into a new collection tube The yield can be improved by an additional 20 30 when this second elution is performed 5 Storage of DNA The purified DNA sample may be stored at 4 C for a few days It is recommended that samples be placed at 20 C for long term storage Appendix A Sample Preparation for 0 3 1 mL of Blood Containing Gram Positive Bacterial Pathogens a b C Add 0 1 mL of Lysozyme to a microcentrifuge tube and transfer 0 3 mL 1 mL of blood sample to the tube containing Lysozyme Mix well by vortexing and then incubate at 37 C for 1 hour 0 5 and 2 hours can be used depending on the bacterial strain being lysed After incubation add 0 1 mL of Proteinase K vortex before use to the tube and proceed to step 1c of Procedure A Isolation of DNA from 0 3 1 mL of Blood Appendix B Sample Preparation for 1 2 mL of Blood Containing Gram Positive Bacterial Pathogens a Add 0 2 mL of Lysozyme to a microcentrifuge tube and transfer 1 mL 2 mL of blood sample to the tube containing Lysozyme b Mix well by vortexing and then incubate at 37 C for 1 hour 0 5 and 2 hours can be used depending on the bacterial strain being lysed c After incubation add 0 2 mL of Proteinase K vortex before use to the tube and proceed to step 1c of Pr
11. ocedure B Isolation of DNA from 1 2 mL of Blood Related Products Product Blood DNA Isolation Micro Kit 52100 Blood DNA Isolation Mini Kit 46300 Blood DNA Isolation Maxi Kit 31200 Leukocyte RNA Purification Kit 21200 HighRanger 1kb DNA Ladder 11900 UltraRanger 1kb DNA Ladder 12100 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 Troubleshooting Guide Problem Possible Cause Solution and Explanation The spin column is clogged Inefficient cell lysis Check Proteinase K activity Also ensure that correct volume of Lysis Buffer B was added to the blood sample Cell debris may be clogging the column When a high cell number is expected in the blood sample ensure that the optional spin for 2 minutes at 5 000 rpm after the Proteinase K incubation is performed Take the clean supernatant only for the next binding step The sample is too large Too many cells were applied to the column Ensure that Proteinase K and Lysis Buffer B are proportionally added as the blood volume is increased Clogging can be alleviated by centrifuging for a longer period of time until the lysate passes through the column The yield of genomic DNA is low Inefficient
12. oncentration of the Wash Solution A by adding 90 mL of 96 100 ethanol provided by the user to each of the supplied bottles containing the concentrated Wash Solution A This will give a final volume of 128 mL The label on the bottle has a box that may be checked to indicate that the ethanol has been added e Pre heat Elution Solution B at 70 C e For blood containing Gram positive bacterial pathogens prepare a 400 mg mL stock solution approximately 1 7 x 107 units mL of lysozyme as per supplier s instructions e Always vortex the Proteinase K before use A Isolation of DNA from 0 3 1 mL of Blood 1 Sample Preparation NOTE For DNA isolation from blood containing Gram positive bacterial pathogens please see Appendix A for Sample Preparation a Add 0 1 mL of Proteinase K vortex the Proteinase K before use to a 15 mL tube b Transfer 0 3 1 mL of blood sample to the tube containing Proteinase K c Add 1 5 mL of Lysis Buffer B to the blood and mix vigorously by shaking the tube for 10 seconds then vortex vigorously for 10 seconds Note During vortex ensure that the contents are being mixed well d Briefly spin the tube to collect any drops of liquid from the inside of the lid e Incubate at 56 C for 15 minutes mixing once during incubation by vigorous shaking Note If any debris is present in the sample centrifuge for 2 minutes at 4 500 x g 5 000 RPM to precipitate Transfer the clean supernatant to a clean tube
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