E.Z.N.A.®BAC/PAC DNA Kit - Omega Bio-Tek
Contents
1. Let sit at room temperature for 5 minutes Centrifuge at maximum speed for 2 minutes Store DNA at 20 C E Z N A BAC PAC DNA Kit Protocol E Z N A BAC PAC DNA Kit Low Copy Number Protocol Materials and Equipment to be Supplied by User Microcentrifuge capable of at least 18 000 x g Vortexer e Ice bucket Nuclease free 1 5 2 mL microcentrifuge tubes 10 15 mL culture tubes e 70 Ethanol e lsopropanol Sterile Deionized Water or TE Buffer Before Starting e Prepare T1 Buffer BAC Binding Buffer and SPM Wash Buffer according to the Preparing Reagents section on Page 4 e Prepare an ice bucket e Chill T3 Buffer on ice 1 Isolate a single colony from a freshly streaked selective plate and inoculate a culture of 2 5 mL LB or YT medium containing the appropriate antibiotic Incubate for 20 24 hours at 37 C with vigorous shaking 300 rpm Use a flask with a volume at least 4 times the culture volume 2 Centrifuge 1 5 5 mL culture at 3 500 5 000 x g for 3 minutes at room temperature 3 Decant or aspirate and discard the culture media Note To ensure that all traces of the medium are removed use a clean paper towel to blot excess liquid from the wall of the vessel 4 Add 260 uLT1 Buffer RNase A Vortex or pipet up and down to mix thoroughly Complete resuspension of cell pellet is vital for obtaining good yields Note RNase A must be added to T1 Buffer before use Please see the instructions i2. 21 22 23 10 E Z N A BAC PAC DNA Kit Protocol Add 0 7 volumes isopropanol to the samples Vortex at maximum speed for 15 seconds Note For example add 546 uL isopropanol to 780 uL cell lysate Centrifuge at maximum speed for 10 minutes at room temperature Carefully aspirate and discard the supernatant making sure not to dislodge the DNA pellet Add 500 uL 70 ethanol Centrifuge the 2 mL collection tube containing pellet in the same orientation as before for 10 minutes Carefully aspirate and discard the supernatant making sure not to dislodge the DNA pellet Invert the tube containing the DNA pellet on a paper towel for 10 15 minutes to air dry the DNA pellet Note Ensure that no alcohol droplets are visible after air drying Do not over dry the DNA pellet Over drying the DNA pellet will make redissolving the pellet difficult Add 30 50 uL Elution Buffer Let sit overnight at room temperature Store DNA at 20 C Troubleshooting Guide Please use this guide to troubleshoot any problems that may arise For further assistance please contact the technical support staff toll free at 1 800 832 8896 Only use LB or YT medium containing ampicillin Do not use more than 5 mL culture with the standard protocol Cells may not have been dispersed Poor cell lysis adequately prior to the addition of T2 Buffer Make sure to vortex cell suspension to completely disperse Increase incubation time with T2
3. Information The following components are available for purchase separately Call Toll Free at 1 800 832 8896 DNase RNase free microcentrifuge tubes 1 5 mL 500 pk 10 pk cs SSI 1210 00 DNase RNase free microcentrifuge tubes 2 0 mL 500 pk 10 pk cs SSI 1310 00 HiBind E Z N A and MicroElute are registered trademarks of Omega Bio tek Inc PCR is a patented process of Hoffman La Roche Use of the PCR process requires a license 13 Notes
4. into a 2 mL Collection Tube Optional Protocol for Column Equilibration 1 Add 100 uL 3M NaOH to the HiBind DNA Mini Column 2 Centrifuge at maximum speed for 30 60 seconds 3 Discard the filtrate and reuse the collection tube 13 14 iS 16 17 18 19 20 21 22 23 24 E Z N A BAC PAC DNA Kit Protocol Transfer the cleared supernatant from Step 10 to the HiBind DNA Mini Column Be careful not to disturb the pellet and that no cellular debris is transferred to the HiBind DNA Mini Column Centrifuge at maximum speed for 30 seconds Discard the filtrate and reuse the collection tube Add 750 uL SPM Wash Buffer Note DNA Wash Buffer must be diluted with 100 ethanol prior to use Please see Page 4 for instructions Centrifuge at maximum speed for 1 minute Discard the filtrate and reuse the collection tube Centrifuge the empty HiBind DNA Mini Column for 2 minutes at maximum speed to dry the column matrix Note It is important to dry the HiBind DNA Mini Column matrix before elution Residual ethanol may interfere with downstream applications Transfer the HiBind DNA Mini Column to a clean 1 5 mL microcentrifuge tube Add 30 50 uL Elution Buffer or sterile deionized water directly to the center of the column membrane Note The efficiency of eluting DNA from the HiBind DNA Mini Column is dependent on pH If using sterile deionized water make sure that the pH is around 8 5
5. Buffer to Low DNA yields i obtain a clear lysate T2 Buffer if not tightly closed may need to be replaced Bacterial colony is not Use fresh glycerol cultures and avoid fresh repeated freeze thaw cycles Always make enough replica plates and use precultures for inoculation Any remaining precultures can be used to set up fresh glycerol stocks Check the stock of buffers and age of the buffers Make sure that the correct volume of buffer has been added to the sample T2 Butfer precipitated Warm up the T2 Buffer to dissolve the No DNA Eluted ae precipitate Pelleted cells should be completely Cells are not completely resuspended with T1 Buffer Do not add resuspended T2 Buffer until an even cell suspension is obtained Lysate prepared incorrectly 11 Troubleshooting Guide Over mixing of cell High molecular lysate upon addition of weight DNA T2 Buffer contamination Overgrown culture contains lysed cells and of product Culture overgrown degraded DNA Do not grow cell for longer than 16 hours Do not vortex or mix aggressively after adding T2 Buffer DNA degraded High levels of Perform the heat inactivation step after Saal endonuclease ee Problem e lene Solution RNase A not added to T2 Add 1 vial of RNase to each bottle of T2 Buffer Buffer DNA floats out of well while loading agarose gel RNA visible on agarose gel Air dry the DNA pellet before redissolving the DNA 12 Ordering
6. Ca OMEGA Innovations in nucleic acid isolation Product Manual E Z N A BAC PAC DNA Kit D2156 00 5 preps D2156 01 50 preps May 2013 For research use only Not intended for diagnostic testing E Z N A BAC PAC DNA Kit Table of Contents Introduction and OVEFVIEW csccsccsscsecssccssecssecnsecseecseccneersees 2 Kit Contents Storage and Stability sscsecsssecsecseeesers 3 Preparing Reagents seseesssserssseeessseeesseeessseessnseossessssssssssseossss 4 Yield and Quality Of DNA sssssesssssessessessssssrsssssessessessessssssssssss 4 Standardi PrOtOCOlusssissiiciisssestirsssienieirirsarrietiriiacirat siei 5 Low Copy Number Protocol sessssssssecssscssscsessscsssecsecseeessees 8 Troubleshooting Guid iscsi ence ce 11 Ordering snar eerste 13 Manual Revision May 2013 02 OMEGA bio tek Innovations in nucleic acid isolation Introduction and Overview The E Z N A BAC PAC DNA Kit is designed for rapid high throughput purification of BACs PACs and P1s from small volume bacterial cultures This kit is based on a modified alkaline lysis procedure that has been adapted for use with spin columns and high throughput procedures The protocol has been tested using a variety of low copy cosmids BACs PACs P1s and E coli strains In addition this kit can also be used for high copy plasmid isolation Two protocols are provided in this handbook for your convenience The new standard protocol provides a fas
7. Nase A Vortex or pipet up and down to mix thoroughly Com plete resuspension of cell pellet is vital for obtaining good yields Note RNase A must be added to T1 Buffer before use Please see the instructions in the Preparing Reagents section on Page 4 10 11 12 E Z N A BAC PAC DNA Kit Protocol Add 200 uL T2 Buffer Invert and gently rotate the tube 10 20 times to obtain a clear lysate Note Avoid vigorous mixing as this will shear chromosomal DNA and lower BAC purity Do not allow the lysis reaction to proceed more than 5 minutes Store T2 Buffer tightly capped when not in use to avoid acidification from CO in the air Let sit for 5 minutes at room temperature Add 200 uL cold T3 Buffer Immediately invert several times until a flocculent white precipitate forms Note It is vital that the solution is mixed thoroughly and immediately after the addition of T3 Buffer to avoid localized precipitation If the mixture still appears viscous brownish or conglobated more mixing is required to completely neutralize the solution Incubate on ice for 5 minutes Centrifuge at maximum speed for 10 minutes at 4 C Transfer the cleared supernatant to a new 1 5 mL microcentrifuge tube not provided Add 200 uL BAC Binding Buffer Invert the tube 3 5 times to mix thoroughly Note BAC Binding Buffer must be diluted with isopropanol prior to use Please see Page 4 for instructions Insert a HiBind DNA Mini Column
8. dicates greater than 90 nucleic acid Alternatively quantity as well as quality sometimes can be determined best by agarose gel ethidium bromide electrophoresis by comparison to DNA samples of known concentrations Typically the majority of the DNA eluted is in monomeric supercoil form though concatemers may also be present E Z N A BAC PAC DNA Kit Protocol E Z N A BAC PAC DNA Kit Standard Protocol Materials and Equipment to be Supplied by User Microcentrifuge capable of at least 18 000 x g Vortexer e Ice bucket Nuclease free 1 5 2 mL microcentrifuge tubes 10 15 mL culture tubes 70 Ethanol e lsopropanol Sterile Deionized Water or TE Buffer Before Starting e Prepare T1 Buffer BAC Binding Buffer and SPM Wash Buffer according to the Preparing Reagents section on Page 4 e Prepare an ice bucket e Chill T3 Buffer on ice 1 Isolate a single colony from a freshly streaked selective plate and inoculate a culture of 2 5 mL LB or YT medium containing the appropriate antibiotic Incubate for 20 24 hours at 37 C with vigorous shaking 300 rpm Use a flask with a volume at least 4 times the culture volume 2 Centrifuge 1 5 5 mL culture at 3 500 5 000 x g for 3 minutes at room temperature 3 Decant or aspirate and discard the culture media Note To ensure that all traces of the medium are removed use a clean paper towel to blot excess liquid from the wall of the vessel 4 Add 200 uLT1 Buffer R
9. n the Preparing Reagents section on Page 4 10 11 12 13 14 E Z N A BAC PAC DNA Kit Protocol Add 260 uL T2 Buffer Invert and gently rotate the tube 10 20 times to obtain a clear lysate Note Avoid vigorous mixing as this will shear chromosomal DNA and lower BAC purity Do not allow the lysis reaction to proceed more than 5 minutes Store T2 Buffer tightly capped when not in use to avoid acidification from CO in the air Let sit for 5 minutes at room temperature Add 260 uL cold T3 Buffer Immediately invert several times until a flocculent white precipitate forms Note It is vital that the solution is mixed thoroughly and immediately after the addition of T3 Buffer to avoid localized precipitation If the mixture still appears viscous brownish or conglobated more mixing is required to completely neutralize the solution Incubate on ice for 5 minutes Centrifuge at maximum speed for 10 minutes at 4 C Insert a HiBind DNA Mini Column into a 2 mL Collection Tube Transfer the cleared supernatant from Step 10 to the HiBind DNA Mini Column Be careful not to disturb the pellet and that no cellular debris is transferred to the HiBind DNA Mini Column Centrifuge at maximum speed for 1 minute Discard the HiBind DNA Mini Column Do not discard the filtrate Add 2 uL linear polyacrylamides to the 2 mL Collection Tube containing the cleared cell lysate 15 16 17 18 19 20
10. t and reliable method for purification of BAC PAC P1 and plasmid DNA using a HiBind DNA Mini Column The second protocol is for the isolation of low copy number cosmids BACs PACs and P1s New in this Edition e This manual has been edited for content and redesigned to enhance user readability e Equilibration Buffer is no longer included with this kit An optional Column Equilibration Protocol has been added to the protocol for your convenience Equilibration Buffer is replaced with 3M NaOH provided by the user Kit Contents oaa oo o 10 mM Tris HCI pH 8 5 Storage and Stability All of the E Z N A BAC PAC DNA Kit components are guaranteed for at least 12 months from the date of purchase when stored as follows RNase A should be stored at 2 8 C T1 Buffer once RNase A is added should be stored at 2 8 C All remaining components should be stored at room temperature Preparing Reagents 1 Add the vial of RNase A to bottle of T1 Buffer Store at 4 C 2 Dilute BAC Binding Buffer with isopropanol as follows and store at room temperature 3 Dilute SPM Wash Buffer with 100 ethanol as follows and store at room temperature Yield and Quality of DNA Determine the absorbance of an appropriate dilution 20 to 50 fold of the sample at 260 nm and then at 280 nm The DNA concentration is calculated as follows DNA concentration Absorbance 260 x 50 x Dilution Factor pg mL A ratio greater than 1 8 in
Download Pdf Manuals
Related Search