Plasmid Fast Midiprep Kit
Contents
1. from low storage temperatures and dissolve the SDS by warming DNA is found in the flow through of cleared lysate a Plasmid Purification membrane overloaded b RNase A digestion omitted c RNase A digestion Insufficient d SDS was in lysate No DNA in eluate a No DNA in the lysate b DNA passed through in the flow through or wash Plasmid Fast Midiprep Kit If rich culture media such as TB or 2x YT are used culture volumes must be reduced It may be necessary to adjust LB culture volume if the plasmid and host strain show extremely high copy number or growth rates Ensure that RNase A is added to Buffer T1 before use Reduce culture volume if necessary If Buffer T1 containing RNase A is more than 6 months old add additional RNase A Buffer T3 before use If lysate is too viscous for effective mixing of Buffer T3 reduce culture volume or increase volumes of Buffer T1 T2 and T3 See section No DNA in lysate See previous two sections m biotech messenger of biotechnology fraction Little or no DNA after precipitation a DNA failed to precipitate b DNA pellet was lost c DNA was poorly redissolved Ensure that the precipitate is centrifuged at 215 000 x g for 30 min Recover DNA by centrifuging for longer and at higher speeds Try another isopropanol batch Isopropanol pellets are glassy and may be difficult to see Mark the outside of the tube before centrifugation Isoprop2. DNA poor quality DNA a Genomic DNA in the eluate b RNA in the eluate Plasmid Fast Midiprep Kit Mixing of bacterial lysate was too vigorous The lysate must be handled gently after additional Buffer T1 T2 and T3 to prevent shearing of chromosomal DNA Reduce culture volume if lysate is too viscous for gentle mixing RNase A digestion was insufficient Check culture volume against recommended volumes and reduce if necessary Check that the RNase A provided with the kit has been used m biotech messenger of biotechnology c Nuclease contamination d Lysis time was too Long e Overloaded alkaline lysis f Plasmid DNA is nicked sheared Degraded g Shearing during Redissolving h Particles in redissolved DNA Poor DNA performance a Too much Salt in pellet b Residual protein If Buffer T1 is more than 6 months old add more RNase A Check buffers for necessary Use new glass and plasticware and wear gloves nuclease contamination and replace if Ensure that lysis step Buffer T2 does not exceed 5 min Check the culture volume and yield against the capacity of the Spin Column Reduce the culture volume accordingly or alternatively increase the volume of Buffer Ti T2 and T3 DNA was poorly buffered Redissolve DNA in Buffer TE pH 8 0 to inhibit nuclease activity and maintain stable pH during storage Redissolve DNA gently without vortexing or vigorous pipetting Avoid using smal
3. Plasmid Fast Midiprep Kit High quality Plasmid Purification Kits for Transfection User Manual Version 6 0 Catalog No 11510F 11520F 11500F Storage Conditions Room Temperature For Research Use Only www mbiotech co kr i Principle The m biotech High quality Plasmid Purification Kits for Transfection procedure is based on a modified alkaline lysis method followed by binding of plasmid DNA onto membrane in the presence of low salt and pH conditions RNA proteins dyes and low molecular weight impurities are removed by a medium salt wash Plasmid DNA is eluted in a high salt buffer and the concentrated and desalted by isopropanol precipitation No expensive equipment such as ultracentrifuges and HPLC or toxic reagents such as phenol and ethidium bromide are required Culture volume Do not exceed the maximum recommended culture volumes given at the beginning of each protocol Using larger culture volumes will lead to an increase in biomass and can affect the efficiency of alkaline lysis leading to reduced yield and purity of the preparation The protocol for High quality Plasmid Purification Kits for Transfection is optimized for use with cultures grown in standard Luria Bertani LB medium grown to a cell density of approximately 3 4 x 109 cells per ml We advise harvesting cultures after approximately 12 16 hours of growth which typically is the transition from logarithmic into stationary growth phase At is best
4. anol pellets may also be loosely attached to the side of the tube so pour supernatant off gently Check that DNA is completely redissolved Be sure to wash any DNA off the walls particularly if glass tubes and a fixed angle rotor are used Up to half of the total DNA may be smeared on the walls Alternatively a swinging bucket rotor can be used to ensure that the pellet is located at the bottom of the tube Plasmid DNA difficult to redissolve a Pellet was overdried b Residual isopropanol in pellet c Too much salt in pellet d Buffer pH was too low e Resuspension Tt volume too low Air dry pellet instead of using a vacuum especially if the DNA is of high molecular weight Redissolve DNA by warming the solution slightly and allowing more time for redissolving Ensure that pellets are washed with 70 ethanol to remove traces of isopropanol Redissolve DNA by warming the solution slightly and allowing more time for redissolving Increase volume of buffer used for redissolving if necessary Ensure that isopropanol is at room temperature for precipitation and wash the pellet twice with room temperature 70 ethanol Recover DNA by increasing the volume of buffer used for redissolving Ensure that the pH of the buffer used for redissolving is 28 0 since DNA does not dissolve well in acidic solutions Increase resuspension T1 volume if the solution above the pellet is highly viscous Contaminated
5. ar gloves when handling these buffer e Isopropanol and 70 ethanol e In Spin Column If remainder should occur Plasmid Fast Midiprep Kit 2 mMm biotech e messenger of biotechnology Protocol for Plasmid Fast Midiprep Kit Reusable High quality Plasmid Purification Kits for Transfection Please read Important Notes before starting Cat No 11510F 11520F 11500F Storage 10 preps 25 prep 100 preps Condition Buffer EQ 50 ml 125 ml 250 mIX 2ea RT Buffer T1 50 ml 125 ml 125 ml X 4ea RT Buffer T2 50 ml 125 ml 250 ml X 2ea RT Buffer T3 50 ml 125 ml 250 ml X 2ea RT Buffer TU 125 ml 125 ml X 2ea 250 ml X 4ea RT 60 ml X1 125 mI X lea Buffer EN 50 ml 125 ml 250 ml X 2ea RT Buffer TE 5 ml 12 ml 60 ml RT RNase A 25mg ml 250 ul 600 ul 600 ul X 4ea 4 Midi Column amp Tube Each 10 ea Each 25 ea Each 100 ea RT Protocol 1 ea lea 1 ea Plasmid Fast Midiprep Kit 3 i we 1 Harvest a 50 ml cultured high copy number plasmids or cosmids gt 2 ug DNA ml LB If you wish to stop the protocol and continue later freeze the cell pellets at 20 C 2 Apply 5 ml of Buffer EQ to the Midi Column Let stand for 1 min Centrifuge at 4 000 x g in a swing bucket rotor at room temperature for 1 min or vacuum OR centrifuge at 8 000 x g in a fixed angle rotor for 3 min disregard of remainder next step Discard the flow through 3 Suspend pelleted bacterial cells in 5 ml of Buffer T1 4 Add 5 ml of Buffe
6. b Precipitator Midi Use the size of Precipitator corresponding to the Spin Column Module was used for being used precipitation of eluate from a column c Ethanol was used for Use of ethanol instead of isopropanol for precipitation leads to a precipitation instead finer precipitate that can clog the module of isopropanol Precipitator casing breaks causing leakage a Excessive exposure of Prolonged incubation with alcohol may weaken the joint between Precipitator to alcohol upper and Jower part of the Precipitator Complete steps 15 20 within 10 15 min b Precipitator attached Do not apply excessive force bending or twisting when attaching with excessive force the Precipitator to the syringe c Precipitator inlet was Do not stress the inlet by resting one edge of the Precipitator on bent during processing a hard surface e g the edge of a sink and depressing the syringe plunger Always apply gentle even pressure perpendicularly to the Precipitator Plasmid Fast Midiprep Kit 10 mMm biotech e messenger of biotechnology Customer amp Technical Services For technical assistance and more information please contact us www mbiotech co kr Tel 82 31 556 3905 Fax 82 31 790 0079 E mail info mbiotech co kr Plasmid Fast Midiprep Kit 11
7. e poured disrupt the precipitate into tiny particles which may clog the Cartridge Pour the lysate into the Cartridge immediately after addition of Neutralization Solution and do not agitate during the 10 min incubation Agitation causes the precipitate to be disrupted into tiny particles instead of forming a layer Ensure incubation is performed at room temperature Buffer T3 on ice instead of in the Cartridge Precipitate flotation is more efficient at room temperature than on ice Incubate with Buffer T3 as indicated in the protocol If the precipitate has not risen to the top after the 10 min incubation carefully run a sterile pipet tip or sterile spatula around the cartridge wall to dislodge the precipitate before continuing with the filtration m biotech messenger of biotechnology Lysate not clear after filtration a Precipitate was forced Filter until all of the lysate has passed through the Midi or Maxi through the Cartridge but do not apply extreme force Approximately 12 ml Catridge Midi or 25 ml Maxi of the lysate are typically recovered Precipitator Modules DNA does not perform well a Eluate contains Ensure that the membrane is dried by pressing air through the residual alcohol Precipitator at least twice Dry the outlet nozzle of the Precipitator with absorbent paper Precipitator clogs during use a Too much DNA applied Do not load eluate from several columns on the Precipitator to the Precipitator
8. ge at 15 000 x g at 4 C for 30 min carefully discard the supernatant 11 Wash DNA pellet with 2 ml 70 ethanol Centrifuge at gt 15 000 x g at 4 C for 10 min Carefully and fully pipet off the ethanol wash without disturbing the pellet Air dry the pellet 12 Redissolve the DNA in 0 4 ml or a suitable volume_of Buffer TE pH 8 0 or 10 mM Tris Cl pH8 5 Plasmid Fast Midiprep Kit 5 mM biotech e messenger of biotechnology Troubleshooting Guiade This troubleshooting guide may be helpful in solving any problems that may arise Problem Comments and suggestions Low or no DNA yield No DNA in lysate a Plasmid did not Propagate b Alkaline lysis was Inefficient c Insufficient lysis for low copy plasmids d Lysate incorrectly prepared Check the conditions for optimal growth If cells have grown to very high densities ora larger amount of cultured medium than recommended was used the ratio of biomass to lysis reagent is shifted This may result in poor lysis conditions because the volumes of Buffer T1 T2 and T3 are not sufficient for setting the plasmid DNA free efficiently Reduce culture volume or increase volumes of Buffer T1 T2 and T3 Also insufficient mixing of lysis reagents will result in reduced yield For low copy plasmid preparations doubling the volumes of Buffers T1 T2 and T3 may help to increase plasmid yield and quality Check Buffer T2 for SDS precipitation resulting
9. ion analysis Cosmid clones in particular should always be prepared from freshly streaked well isolated colonies since cosmids are not stable in E co i for long periods of time Ensure that the lysate is clear before it is loaded onto the column Ensure that Buffer T3 is chilled before use Check g force and centrifugation time Alternatively clear the lysate using a Cartridge To clear a blocked Spin Column positive pressure may be applied e g by using a syringe fitted into a rubber stopper with a hole Cartridges Cartridge clogs during filtration a Too large culture volume used b Inefficient mixing after addition of Buffer T3 c Mixing after addition of Buffer T3 too vigorous d Cartridge not immediately after was loaded addition of Buffer T3 e Cartridge was agitated during incubation f Incubation after addition of Buffer T3 on ice instead of at RT g Incubation time after addition of Buffer T3 too short Plasmid Fast Midiprep Kit Use no more than the culture volume recommended in the protocol Mix well until a fluffy white material has formed and the lysate is no longer viscous After addition of Buffer T3 the immediately but gently Vigorous mixing disrupts the precipitate lysate should be mixed into tiny particles which may clog the Cartridge After addition of Buffer 13 the immediately into the Cartridge Decanting after incubation may lysate should b
10. l pipet tips Centrifuge the DNA solution and transfer supernatant to a new tube The particles have no affect on DNA quality Alternatively use UltraHiFast kits containing Precipitatior which filters the eluate Ensure that isopropanol is at room temperature for precipitation and wash the pellet twice with room temperature 70 ethanol Precipitate the DNA again to remove the salt Check culture volume against the recommended volumes and reduce if necessary Ensure that the bacterial lysate is cleared properly by centrifugation at 215 000 x g for 45 min or using a Cartridge Extra DNA bands on analytical gel a Dimer form of plasmid b Plasmid has formed Denatured supercoils Plasmid Fast Midiprep Kit Dimers or multimers of supercoiled plasmid DNA are formed during replication of plasmid DNA Typically when purified plasmid DNA is electrophoresed both the supercoiled monomer and dimer form of the plasmid are detected upon ethidium bromide staining of the gel The ratio of these forms is often host dependent This species runs faster than closed circular DNA on a gel and is resistant to restriction digestion Do not incubate cells for longer than 5 m biotech messenger of biotechnology c Possible deletion mutants Blocked Spin Column a Lysate was turbid min in Buffer T2 Mix immediately after addition of Buffer T3 Some sequences are poorly maintained in plasmids Check for deletions by restrict
11. r T2 Mix gently by inverting the capped tube five times Do not vortex Incubate at room temperature for 5 min 5 Add 5 ml of Buffer T3 Mix immediately by inverting the tube five times Do not vortex 6 Centrifuge at 15 000 x g at room temperature for 10 min If centrifugation is done at 4 C supernatant must be warmed to room temperature before loading on column 7 Apply the cleared lysate of step 6 into the equilibrated Midi Column Centrifuge at 4 000 x g in a swing bucket rotor at room temperature for 1 min or vacuum OR centrifuge at 8 000 x g in a fixed angle rotor for 3 min disregard of remainder next step Discard the flow through 8 Apply 12 ml of Buffer TU to the Midi Column Centrifuge at 4 000 x g in a swing bucket rotor at room temperature for 2 min or vacuum OR centrifuge at 8 000 x g in a fixed angle rotor for 3 min disregard of remainder next step Discardthe flow through 9 Place the Midi Column in a clean 50 ml centrifuge tube not provided Apply 5 ml of Buffer EN to the Midi Column Centrifuge at 4 000 x g in a swing bucket rotor at room temperature for 2 min OR centrifuge at 28 000 x g in a fixed angle rotor for 3 min disregard of remainder next step Note For constructs larger than 45 50 kb prewarming the Buffer EN to 65 C may help to increase yield Plasmid Fast Midiprep Kit 4 10 Precipitate DNA by adding 0 7 volume isopropanol to the eluted DNA Mix and centrifu
12. to assess the cell density of the culture and if that is too high reduce the culture volumes accordingly or increase the volumes of Buffer T1 Buffer T2 Buffer T3 A high ratio of biomass to lysis buffers will result in poor lysis conditions and subsequently low DNA yield and purity For determination of cell density calibration of each individual spectrophotometer is required to facilitate accurate conversion of OD600 measurements into the number of cells per ml This can be achieved by plating serial dilutions of a culture onto LB agar plates in the absence of antibiotics The counted colonies are used to calculate the number of cells per ml which is then set in relation to the measured OD600 values Convenient stopping points in protocols The purification procedure can be stopped and continued later by freezing the cell pellets obtained by centrifugation The frozen cell pellets may be stored at 20 C for several weeks In addition the DNA eluted from the Spin or Ez Column may be stored overnight at 2 8 C after which the protocol can be continued Important Note Please read the following notes before starting any of the Plasmid Purification procedures for Transfection Plasmid Fast Midiprep Kit 1 mM biotech e messenger of biotechnology Before equipment e Add the RNase A Solution to Buffer T1 mix store at 2 87 e Check Buffer T2 before use for salt precipitation If any precipitated heat to dissolve 37 C We
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