plasmid dna puri˜cation - geneaid · plasmid dna purification page 2 figure 2. geneaid pdh columns...

Plasmid DNA Pur i�cat ion

Presto TM M ini Plasmid K it . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .1Presto TM Endotoxin Free M ini Plasmid K it . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .2H igh-Speed Plasmid M ini K i t . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .3Large Plasmid DNA Ex trac t ion K it . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .4H igh-Speed Plasmid Advance K it (50-100 ml) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .5Presto TM M idi Plasmid K it . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .6Presto TM M idi Plasmid K it (Endotoxin Free) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .7Geneaid TM M idi Plasmid K it . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .8Geneaid TM M idi Plasmid K it (Endotoxin Free) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .9Presto TM Plasmid DNA Concentrat ion K it . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .10Geneaid TM Maxi Plasmid K it . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .11Geneaid TM Maxi Plasmid K it (Endotoxin Free) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .12Presto TM 96 Wel l P lasmid K it . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .13Presto TM Plasmid 96 Wel l B inding Plate. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .14Presto TM Plasmid 96 Wel l Fi l ter Plate. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .14

Plasmid DNA PurificationPage 1 www.geneaid.com

Figure 3. Geneaid PDH Columns are specially designed and pretreated to ensure high plasmid DNA yields from a wide variety of bacterial strains and sample volumes.

DNA binding to membrane while contaminants remain suspended

Wash (removal of contaminants while DNA remains bound to membrane)

Elution of pure plasmid DNA which is ready for subsequent reactions

Lyse bacterial cells (optional color indicator will turn blue when lysis is successful)

Neutralize suspension (optional color indicator will become clear when neutralization is successful)

Figure 2. Sequencing data pBluescript plasmid DNA purified using the Presto™ Mini Plasmid Kit.

Presto™ Mini Plasmid Kit Test Data

Figure 1. Plasmid DNA was extracted using the Presto™ Mini Plasmid Kit. 5 µl aliquots of a 100 µl eluate of purified super coiled plasmid DNA from 1.5, 3, 5 and 7 ml overnight E. coli (DH5α) culture, containing a 3 kb plasmid pBluescript and pBR322 (OD600 = 4 U/ml) were used in EcoRI digestion and analyzed by electrophoresis on a 0.8% agarose gel. M = Geneaid 1 Kb DNA LadderpBluescript: 1=1.5 ml, 2=3 ml, 3=5 ml, 4=7 mlpBR322: 5=1.5 ml, 6=3 ml 7=5 ml, 8=7 ml

1 2 3 4 M 5 6 7 8

Copy Number Host Strain Cell Culture Volume (OD600 = 4.0)

1.5 ml 3 ml 5 ml 7 ml

High-Copy (pBluescript)

13-15 µg 27-29 µg 36-38 µg 40-42 µg

Low-Copy (pBR322)

4-6 µg 8-10 µg 12-14 µg 18-20 µg

Advantages• Purify plasmid DNA within 15 minutes!• TrueBlue Lysis Buffer is an optional color indicator included with the kit to reduce common handling errors, while ensuring efficient cell lysis, neutralization and maximum plasmid product yield• High Yield: up to 50 µg of pure plasmid DNA• 1-7 ml of cultured bacterial cells (1-15 kb plasmid)• Elution Volume: 30-100 µl • Plasmid glass fiber spin column • Storage: dry at room temperature (15-25ºC)

ApplicationsRestriction Enzyme Digestion, Library Screening, Ligation, PCR, Transformation and Sequencing Reactions

Components• PD1, PD2, PD3 Buffers• TrueBlue Lysis Buffer• W1 Buffer and Wash Buffer• Elution Buffer (10 mM Tris-HCl, pH8.5 at 25°C)• RNase A• PDH Columns• 2 ml Collection Tubes

Presto™ Mini Plasmid Kit Protocol Procedure

Harvest cultured bacterial cells by centrifuge to form a cell pellet, followed by resuspension

Presto TM M ini Plasmid K it

Introduction (PDH100, PDH300)The Presto™ Mini Plasmid Kit was designed for rapid isolation of plasmid DNA from 1-7 ml of cultured bacterial cells. A modified alkaline lysis method and RNase treatment are used to obtain clear cell lysate with minimal gDNA and RNA contaminants. In the presence of chaotropic salt, plasmid DNA in the lysate binds to the glass fiber matrix of the spin column. Contaminants are removed with a Wash Buffer (containing ethanol) and the purified plasmid DNA is eluted by a low salt Elution Buffer, TE or water. DNA phenol extraction or alcohol precipitation is not required.

www.geneaid.comPlasmid DNA Purification

Figure 2. Geneaid PDH Columns are specially designed and pretreated to ensure high plasmid DNA yields from a wide variety of bacterial strains and sample volumes.

Presto™ Endotoxin Free Mini Plasmid Kit Test Data

Advantages• Transfection Grade Plasmid DNA: <0.1 EU/µg of plasmid DNA• TrueBlue Lysis Buffer is an optional color indicator included with the kit to reduce common handling errors, while ensuring efficient cell lysis, neutralization and maximum plasmid product yield• High Yield: up to 40 µg of pure plasmid DNA• 1-5 ml of cultured bacterial cells (1-15 kb plasmid)• Plasmid glass fiber spin column• Elution Volume: 30-100 µl • Storage: dry at room temperature (15-25ºC)


Components• PE1, PE2, PE3, PE4, PE5 Buffers• TrueBlue Lysis Buffer• W1 Buffer and Wash Buffer• Elution Buffer (10 mM Tris-HCl, pH8.5 at 25°C)• RNase A• PDH Columns• 2 ml Collection Tubes

Presto™ EF Mini Plasmid Kit Protocol Procedure

Presto TM Endotoxin Free M ini Plasmid K it

Endotoxin free DNA binding to membrane while contaminants remain suspended


Elution of pure Endotoxin Free plasmid DNA which is ready for subsequent reactions




Endotoxin is precipitated in the red pellet followed by endotoxin removal

Figure 1. Plasmid DNA was extracted using the PrestoTM Endotoxin Free Mini Plasmid Kit from a 4 ml of overnight cultured E. coli strain DH5α harboring pBluescript. 2 μl aliquots of a 50 μl eluate of purified super coiled plasmid were analyzed by spectrophotometer and 0.8% agarose gel. M = Geneaid 1 Kb DNA Ladder

Sample ng/µl 260/280 260/230 Yield (ng) 1 569.4 1.89 2.32 28.5 2 578.4 1.89 2.30 28.9 3 594.4 1.89 2.31 29.7

M 1 2 3

Introduction (PEH100, PEH300)The Presto™ Endotoxin Free Mini Plasmid Kit was designed for transfection grade plasmid DNA purification from 1-5 ml of cultured E. coli cells using silica-membrane spin columns. A modified alkaline lysis method and RNase treatment are used to obtain clear cell lysate with minimal genomic DNA and RNA contaminants. In addition, an efficient endotoxin removal step is integrated in the procedure and endotoxins are precipitated in a proprietary reagent. TrueBlue Lysis Buffer (an optional color indicator) is included with the kit in order to prevent common handling errors, ensuring efficient cell lysis and neutralization. DNA phenol extraction or alcohol precipitation is not required and the entire procedure can be completed within 50 minutes. The purified endotoxin free plasmid DNA is ready for use in transfection, restriction enzyme digestion, ligation, PCR, and sequencing reactions.

www.geneaid.com

Advantages• Purify plasmid DNA within 15 minutes!• Up to 30 µg of pure plasmid DNA from 1-5 ml of cultured bacterial cells• Plasmid glass fiber spin column • Elution Volume: 30-100 µl • Storage: dry at room temperature (15-25ºC)


Components• PD1, PD2, PD3 Buffers• W1 Buffer• Wash Buffer• Elution Buffer (10 mM Tris-HCl, pH8.5 at 25°C)• RNase A• PD Columns• 2 ml Collection Tubes

Figure 1. Plasmid DNA was extracted using both the High-Speed Plasmid Mini Kit (lane 3,4) and the equivalent competitor's plasmid kit (lane 1,2). The purified supercoiled plasmid DNA [1.5 ml overnight E. coli (DH5α) culture, containing a 3 kb plasmid pBluescript (A600 > 2 U/ml)], from a 50 µl eluate was used in EcoRI digestion and analyzed by electrophoresis on a 1% agarose gel. M = Geneaid 1 Kb DNA Ladder

Kit Test Yield 260/280 260/230

Competitor 1 8.30 µg 1.88 2.25

2 8.40 µg 1.88 2.24

Geneaid 3 8.30 µg 1.87 2.21

4 8.50 µg 1.89 2.23

1 2 M 3 4

High-Speed Plasmid Mini Kit Test Data

Figure 2. Sequencing data of (pBluescript) plasmid DNA purified using the High-Speed Plasmid Mini Kit.

High-Speed Plasmid Mini Kit Protocol Procedure

Figure 3. Geneaid PD Columns are specially designed and pretreated to ensure high plasmid DNA yields from a wide variety of bacterial strains and sample volumes.





Neutralize suspension

H igh-Speed Plasmid M ini K i t

Plasmid DNA PurificationPage 3

Introduction (PD100, PD300)The High-Speed Plasmid Mini Kit is was designed for plasmid and cosmid DNA purification from 1-5 ml of cultured bacterial cells. A modified alkaline lysis method and RNase treatment are used to obtain clear cell lysate with minimal genomic DNA/RNA contaminants. In the presence of chaotropic salt, plasmid DNA in the lysate binds to the glass fiber matrix of the spin column. Contaminants are removed with a Wash Buffer (containing ethanol) and the purified plasmid DNA is eluted by a low salt Elution Buffer, TE or water. Typical yields are 20-30 μg for high-copy plasmid or 3-10 μg for low-copy plasmid from 4 ml of cultured bacterial cells. DNA phenol extraction or alcohol precipitation is not required.

Plasmid DNA Purification www.geneaid.com

M 1 2 3

Figure 1. Plasmid DNA was extracted using the Large Plasmid DNA Extraction Kit. 5 µl aliquots of a 100 µl eluate of purified super coiled plasmid DNA from a 4 ml overnight E. coli (DH5α) culture, containing a 17.4 kb low copy plasmid pBluescript (OD600 = 3.5 U/ml) were used in EcoRI digestion and analyzed by electrophoresis on a 0.8% agarose gel.M = Geneaid 1 Kb DNA Ladder

Large Plasmid DNA Extraction Kit Test Data

Advantages• Purify 10-50 kb plasmid DNA within 20 minutes!• High Yield: up to 30 µg of pure plasmid DNA• 1-4 ml of cultured bacterial cells• Plasmid glass fiber spin column • Elution Volume: 50-100 µl • Storage: dry at room temperature (15-25ºC)


Components• PDL1 Buffer• PDL2 Buffer • PDL3 Buffer• W1 Buffer• Wash Buffer• Elution Buffer (10 mM Tris-HCl, pH8.5 at 25°C)• RNase A• LP Columns• 2 ml Collection Tubes

Large Plasmid DNA Extraction Kit Protocol Procedure

Figure 2. Geneaid LP Columns are specially designed and pretreated to ensure high 10-50 kb plasmid DNA yields from a wide variety of bacterial strains and sample volumes.





Neutralize suspension

Large Plasmid DNA Ex trac t ion K it

Test Conc. 260/280 260/230 Yield 1 87.0 ng/µl 1.90 2.16 8.7 µg 2 79.0 ng/µl 1.90 2.14 7.9 µg 3 86.4 ng/µl 1.89 2.17 8.6 µg

Introduction (PDL100, PDL300)The Large Plasmid DNA Extraction Kit was designed for plasmid and cosmid DNA purification from 1-4 ml of cultured bacterial cells. The kit is optimized for purification of DNA between 10-50 kb. A modified alkaline lysis method and RNase treatment are used to obtain clear cell lysate with minimal genomic DNA and RNA contaminants. In the presence of chaotropic salt, plasmid DNA in the lysate binds to the glass fiber matrix of the spin column. Contaminants are removed with a Wash Buffer (containing ethanol) and the purified plasmid DNA is eluted by a low salt Elution Buffer, TE or water. Typical yields are 20-30 μg for high-copy plasmid or 3-10 μg for low-copy plasmid from 4 ml of cultured bacterial cells. DNA phenol extraction or alcohol precipitation is not required.

www.geneaid.com

Advantages• Purify plasmid DNA within 30 minutes!• High Yield: up to 500 µg of pure plasmid DNA• 50-100 ml of cultured bacterial cells• Plasmid glass fiber spin column • Elution Volume: 2 ml • Storage: dry at room temperature (15-25ºC)


Components• PD1, PD2, PD3 Buffers• W1 Buffer• Wash Buffer• Elution Buffer (10 mM Tris-HCl, pH8.5 at 25°C)• RNase A• PA Columns

Midiprep Spin Column Plasmid Kit Protocol Procedure

Figure 2. Geneaid PA Columns are specially designed to ensure high plasmid DNA yields from 50-100 ml of cultured bacterial cells.




Harvest cultured bacterial cells by centrifuge to form a cell pellet

Resuspend bacterial cell pellet and neutralize suspension

Figure 1. Plasmid DNA was extracted using the Midiprep Spin Column Plasmid Kit. The purified supercoiled plasmid DNA [50 ml overnight E. coli (DH5α) culture, containing a 3 kb plasmid pBluescript (A600 > 2 U/ml)], from a 2 ml eluate was used in EcoRI digestion and analyzed by electrophoresis on a 1% agarose gel. M = Geneaid 1 Kb DNA LadderLane 1-3: pBluescript II

Midiprep Spin Column Plasmid Kit Test Data

M 1 2 3

M idiprep Spin Column Plasmid K it

Plasmid DNA PurificationPage 5

Introduction (PA025)The Midiprep Spin Column Plasmid Kit was designed for plasmid and cosmid DNA purification from 50-100 ml of cultured bacterial cells. A modified alkaline lysis method and RNase treatment are used to obtain clear cell lysate with minimal genomic DNA/RNA contaminants. In the presence of chaotropic salt, plasmid DNA in the lysate binds to the glass fiber matrix of the spin column. Contaminants are removed with a Wash Buffer (containing ethanol) and the purified plasmid DNA is eluted by a low salt Elution Buffer, TE or water. Typical yields are 200-350 μg for high-copy plasmid or 30-100 μg for low-copy plasmid from 50 ml of cultured bacterial cells. DNA phenol extraction or alcohol precipitation is not required.

Plasmid DNA Purification www.geneaid.com

Advantages• High Yield: >400 µg of pure transfection grade plasmid DNA from 100 ml of cultured bacterial cells• TrueBlue Lysis Buffer (an optional color indicator) is included with the kit to reduce common handling errors, while ensuring efficient cell lysis, neutralization and maximum plasmid product yield• 50-150 ml of cultured bacterial cells• Elution Volume: 500 µl ~ 2 ml• Storage: dry at room temperature (15-25ºC) • Time: only 40 minutes when combined with the Presto™ Plasmid DNA Concentrator (Page 10)

ApplicationsTransfection, Sequencing, Ligation, PCR, In-vitro Transcription, Microinjection, Restriction Enzyme Digestion, Gene Gun

Components• PM1, PM2, PM3 Buffers• TrueBlue Lysis Buffer• PEQ Buffer• PW Buffer• PEL Buffer• RNase A• Filter Columns (yellow spin columns)• Plasmid Midi Columns (anion exhange silica resin columns)

Presto™ Midi Plasmid Kit Protocol Procedure

Presto™ Midi Plasmid Kit Test Data

1 2 M 3 4

Kit Test 260/280 260/230 Total Yield

Geneaid 50 ml 1.85 2.25 273.00 µg

100 ml 1.87 2.14 409.20 µg

MN 50 ml 1.85 2.26 121.10 µg

100 ml 1.87 2.33 289.80 µg

Figure 1. Plasmid DNA was extracted using both the Presto™ Midi Plasmid Kit (lane 1, 2) and the equivalent competitor's plasmid midi kit (lane 3, 4). The purified supercoiled plasmid DNA [50 ml and 100 ml overnight E. coli (DH5α) culture, containing a 3 kb plasmid pBluescript (A600 > 2 U/ml, OD600 = 3.8)], was used in EcoRI digestion and analyzed by electrophoresis on a 1% agarose gel.

M = Geneaid 1 Kb DNA Ladder, Lane 1: Presto™ Midi Plasmid Kit (50 ml), Lane 2: Presto™ Midi Plasmid Kit (100 ml), Lane 3: Equivalent Competitor Kit (50 ml), Lane 4: Equivalent Competitor Kit (100 ml)

Figure 2. The Presto™ Midi Column was designed for centrifuge filtration. The Plasmid Midi Column was designed to ensure high plasmid DNA yields from a wide variety of bacterial strains.

Wash (removal of contaminants while DNA remains bound to silica resin)

Elution and precipitation of pure plasmid DNA which is ready for subsequent reactions

The Presto™ Midi Column is used with centrifugation to filter the neutralized colorless mixture and remove contaminants to facilitate DNA binding to the silica resin of the Plasmid Midi Column




DNA binding to silica resin while contaminants remain suspended

Presto TM M idi Plasmid K it

Introduction (PIF025)The Presto™ Midi Plasmid Kit was designed for plasmid DNA purification from 50-150 ml of cultured bacterial cells. TrueBlue Lysis Buffer (an optional color indicator) is included with the kit to prevent common handling errors, while ensuring efficient cell lysis and neutralization. A modified alkaline lysis method and RNase treatment are used to obtain clear cell lysate with minimal genomic DNA/RNA contaminants. Using an efficient gravity-flow procedure, plasmid DNA in the crude lysate is bound by the plasmid midi column and the contaminants are removed with PW Buffer. The purified plasmid DNA is eluted by a high salt buffer then precipitated with isopropanol for desalting. Due to the high efficiency of the new Presto™ Midi Spin Column, the entire procedure can be completed in 80 minutes without ultracentrifuges, HPLC or other toxic reagents.

Plasmid DNA Purificationwww.geneaid.com

Advantages• High Yield: >400 µg of pure transfection grade plasmid DNA from 100 ml of cultured bacterial cells• Efficient endotoxin removal (<0.1 EU/µg DNA) verified by LAL • TrueBlue Lysis Buffer (an optional color indicator) is included with the kit to reduce common handling errors, while ensuring efficient cell lysis, neutralization and maximum plasmid product yield• 50-150 ml of cultured bacterial cells• Elution Volume: 500 µl ~ 2 ml• Storage: dry at room temperature (15-25ºC)


Components• PM1, PM2, PM3 Buffers• TrueBlue Lysis Buffer• PEQ Buffer• PW Buffer• PEL Buffer• PER Buffer• RNase A• Filter Columns (yellow spin columns)• Plasmid Midi Columns (anion exhange silica resin columns)

Presto™ Midi Plasmid Kit EF Protocol

Presto™ Midi Plasmid Kit Test Data

1 2 M 3 4

Kit Test 260/280 260/230 Total Yield

Geneaid 50 ml 1.85 2.25 273.00 µg

100 ml 1.87 2.14 409.20 µg

MN 50 ml 1.85 2.26 121.10 µg

100 ml 1.87 2.33 289.80 µg

Figure 1. Plasmid DNA was extracted using both the Presto™ Midi Plasmid Kit (lane 1, 2) and the equivalent competitor's plasmid midi kit (lane 3, 4). The purified supercoiled plasmid DNA [50 ml and 100 ml overnight E. coli (DH5α) culture, containing a 3 kb plasmid pBluescript (A600 > 2 U/ml, OD600 = 3.8)], was used in EcoRI digestion and analyzed by electrophoresis on a 1% agarose gel.

M = Geneaid 1 Kb DNA Ladder, Lane 1: Presto™ Midi Plasmid Kit (50 ml), Lane 2: Presto™ Midi Plasmid Kit (100 ml), Lane 3: Equivalent Competitor Kit (50 ml), Lane 4: Equivalent Competitor Kit (100 ml)

Figure 2. The Presto™ Midi Column was designed for centrifuge filtration.The Plasmid Midi Column was designed to ensure high plasmid DNA yields from a wide variety of bacterial strains.



The Presto™ Midi Column is used with centrifugation to filter the neutralized colorless mixture and remove contaminants to facilitate DNA binding to the silica resin of the Plasmid Midi Column



Neutralize suspension (optional color indicator will become clear when neutralization is successful) and endotoxin removal


Presto TM M idi Plasmid K it Endotoxin Free

Introduction (PIFE25)The Presto™ Midi Plasmid Kit Endotoxin Free was designed for plasmid DNA purification from 50-150 ml of cultured bacterial cells. TrueBlue Lysis Buffer (an optional color indicator) is included with the kit to prevent common handling errors, while ensuring efficient cell lysis and neutralization. PER Buffer is included with the kit to ensure efficient endotoxin removal from plasmid preparations. A modified alkaline lysis method and RNase treatment are used to obtain clear cell lysate with minimal genomic DNA/RNA contaminants. Using an efficient gravity-flow procedure, plasmid DNA in the crude lysate is bound by the midi column and the contaminants are removed with PW Buffer. The purified plasmid DNA is eluted by a high salt buffer then precipitated with isopropanol for desalting. Due to the high efficiency of the new Presto™ Midi Spin Column, the entire procedure can be completed in 80 minutes without ultracentrifuges, HPLC or other toxic reagents.


Advantages• High Yield: >400 µg of pure transfection grade plasmid DNA from 100 ml of cultured bacterial cells• Sample: 50-300 ml of cultured bacterial cells• Elution Volume: 500 µl ~ 2 ml• Storage: dry at room temperature (15-25ºC) • Time: only 40 minutes when combined with the Presto™ Plasmid DNA Concentrator (Page 10)


Components• PM1, PM2, PM3 Buffers• TrueBlue Lysis Buffer• PEQ Buffer• PW Buffer• PEL Buffer• RNase A• Plasmid Midi Columns (anion exhange silica resin columns)

GeneaidTM Midi Plasmid Kit Protocol Procedure

Figure 2. The GeneaidTM Midi Plasmid Kit includes the Plasmid Midi Column designed to ensure high plasmid DNA yields from a wide variety of bacterial strains.




Plasmid Midi Kit Test Data

Figure 1. Plasmid DNA from a 100 ml overnight E. coli (DH5α) culture, containing plasmid pBluescript (OD600 = 3.65) was purified using the GeneaidTM Midi Plasmid Kit. The purified supercoiled plasmid DNA was used in EcoRI digestion and analyzed by electrophoresis on a 1% agarose gel.

Test DNA Conc. 260/280 260/230 Yield 1 211.2 µg/ml 1.87 2.27 422.4 µg

2 216.6 µg/ml 1.87 2.27 433.2 µg

3 224.8 µg/ml 1.87 2.28 449.6 µg

M 1 2 3

Geneaid TM M idi Plasmid K it




Introduction (PI025)The GeneaidTM Midi Plasmid Kit was designed for plasmid DNA purification using pre-packed anion-exchange resin columns to purify plasmid DNA from 50-300 ml of cultured bacterial cells. TrueBlue Lysis Buffer (an optional color indicator) is included with the kit to prevent common handling errors, while ensuring efficient cell lysis and neutralization. A modified alkaline lysis method and RNase treatment are used to obtain clear cell lysate with minimal gDNA and RNA contaminants. Using an efficient gravity-flow procedure, plasmid DNA in the crude lysate is bound by the plasmid midi column and the contaminants are removed with PW Buffer. The purified plasmid DNA is eluted by a high salt buffer then precipitated with isopropanol for desalting. The entire procedure can be completed in 2 hours without ultracentrifuges, HPLC or other toxic reagents and the purified plasmid DNA is suitable for Transfection, Sequencing Reactions, Ligation, PCR, In-vitro Transcription, Microinjection, Restriction Enzyme Digestion and Gene Gun.

Test DNA Conc. 260/280 260/230 Yield 1 211.2 µg/ml 1.87 2.27 422.4 µg

2 216.6 µg/ml 1.87 2.27 433.2 µg

3 224.8 µg/ml 1.87 2.28 449.6 µg


Advantages• High Yield: >400 µg of pure transfection grade plasmid DNA from 100 ml of cultured bacterial cells• Efficient endotoxin removal (<0.1 EU/µg DNA) verified by LAL • 50-300 ml of cultured bacterial cells• Elution Volume: 500 µl ~ 2 ml• Storage: dry at room temperature (15-25ºC)


Components• PM1, PM2, PM3 Buffers• TrueBlue Lysis Buffer• PEQ Buffer• PW Buffer• PEL Buffer• PER Buffer• RNase A• Plasmid Midi Columns (anion exhange silca resin columns)

Plasmid Midi Kit EF Test Data

Figure 1. Plasmid DNA from a 100 ml overnight E. coli (DH5α) culture, containing plasmid pBluescript (OD600 = 3.65) was purified using the GeneaidTM Midi Plasmid Kit (EF). The purified supercoiled plasmid DNA was used in EcoRI digestion and analyzed by electrophoresis on a 1% agarose gel.

M 1 2 3

GeneaidTM Midi Plasmid Kit EF Protocol Procedure

Figure 2. The GeneaidTM Midi Plasmid Kit Endotoxin Free includes the Plasmid Midi Column designed to ensure high plasmid DNA yields from a wide variety of bacterial strains.

Wash (removal of contaminants while DNA remains bound to silca resin)






Introduction (PIE25)The GeneaidTM Midi Plasmid Kit Endotoxin Free was designed for plasmid DNA purification using pre-packed anion-exchange resin columns to purify plasmid DNA from 50-300 ml of cultured bacterial cells. TrueBlue Lysis Buffer (an optional color indicator) is included with the kit to prevent common handling errors, while ensuring efficient cell lysis and neutralization. PER Buffer is included with the kit to ensure efficient endotoxin removal from plasmid preparations. A modified alkaline lysis method and RNase treatment are used to obtain clear cell lysate with minimal gDNA and RNA contaminants. Using an efficient gravity-flow procedure, plasmid DNA in the crude lysate is bound by the plasmid midi column and the contaminants are removed with PW Buffer. The purified plasmid DNA is eluted by a high salt buffer then precipitated with isopropanol for desalting. The entire procedure can be completed in 2 hours without ultracentrifuges, HPLC or other toxic reagents and the purified plasmid DNA is suitable for Transfection, Sequencing Reactions, Ligation, PCR, In-vitro Transcription, Microinjection, Restriction Enzyme Digestion and Gene Gun.

Geneaid TM M idi Plasmid K it Endotoxin Free


Plasmid Concentrator DNA Recovery and Concentration

AdvantagesSample: eluate from the Presto™ Midi Plasmid Kit and/or Plasmid Midi KitEfficient: replaces traditional, time-consuming DNA precipitation proceduresBinding Capacity: 600 μg of DNARecovery: up to 90%Concentration: up to 3.2 μg/μlPlasmid Size: 1-20 kb Elution Volume: 100-400 μlDead Volume: 25 μlOperation Time: 10 minutes/prep (manual), 20 minutes/6 preps (vacuum)Storage: dry at room temperature (15-25ºC)

ApplicationsTransfection, Sequencing, Ligation, Library screening, PCR, In-vitro transcription, Microinjection, Restriction enzyme digestion, Gene gun

Components• Presto™ Plasmid Concentrator• Desalting Buffer• Elution Buffer (10 mM Tris-HCl, pH8.5 at 25°C)• 20 ml syringe• 1 ml syringe

Presto™ Plasmid DNA Concentration Kit Protocol Procedure

Elution Volume Input DNA 100 µl 200 µl 400 µl 600 µl

100 µg Recovery 65% 80% 90% 90% Concentration 0.9 µg/µl 0.45 µg/µl 0.2 µg/µl 0.15 µg/µl Eluate 75 µl 175 µl 375 µl 575 µl



Table 1. DNA Recovery is highly dependant on the volume of elution buffer used. 90% recovery can be acheived when using 400 μl of elution buffer. However, DNA concentration is decreased. When using small volumes of elution buffer, recovery is decreased but concentration is increased.

Manual

Precipitate DNA

Load DNA

Add 8 ml of isopropanol to 8 ml of eluate, mix then incubate at RT for 2 mins.

Transfer DNA mixture to a 20 ml syringe.

Press slowly Apply low pressure vacuum until mixture passes throughcompletely

WashAdd 1 ml of Desalting Buffer

Dry Membrane

Elute DNAElute twice for maximum recovery:1. 100-400 μl2. Load first eluate

Press fast > 6X

Press very slowly to elute drop by drop

Press very slowly to elute drop by drop

Turn on vacuum

Vacuum

Presto TM Plasmid DNA Concentrat ion K it

Introduction (PC0250/251, PC0500/501 PC1000/1001)The Presto™ Plasmid DNA Concentration Kit offers a simplified replacement for time-consuming DNA precipitation procedures. The Presto™ Plasmid Concentrator will quickly and efficiently concentrate plasmid DNA in anion-exchange chromatographic DNA purification eluates collected from Geneaid Plasmid Midi Columns, Macherey-Nagel NucleoBond® AX, NucleoBond® Xtra Columns, QIAGEN® tip 500 Columns etc. Plasmid eluates are precipitated with isopropanol then passed through a Presto™ Plasmid Concentrator using a syringe. Once contaminants are removed using a proprietary Desalting Buffer, the concentrator is dried and plasmid DNA is eluted with a small volume (100-400 μl) of low salt Elution Buffer. The Presto™ Plasmid Concentrator eliminates DNA pellet loss and incomplete solubilization of precipitates to ensure highly concentrated, purified plasmid DNA. In only 10 minutes, the plasmid DNA can be used directly in a variety of sensitive downstream applications, such as Transfection, Sequencing, Ligation, Library screening, PCR, In-vitro transcription, Microinjection, Restriction enzyme digestion and Gene gun.


Figure 2. The GeneaidTM Maxi Plasmid Kit includes the Plasmid Maxi Column designed to ensure high plasmid DNA yields from a wide variety of bacterial strains.

GeneaidTM Maxi Plasmid Kit Protocol Procedure

Geneaid TM Maxi Plasmid K it

Advantages• High Yield: 1.5 mg of pure transfection grade plasmid DNA from 400 ml of cultured bacterial cells• 200-1600 ml of cultured bacterial cells• Elution Volume: 500 µl ~ 2 ml• Storage: dry at room temperature (15-25ºC)


Components• PM1, PM2, PM3 Buffers• TrueBlue Lysis Buffer• PEQ Buffer• PW Buffer• PEL Buffer• RNase A• Plasmid Maxi Columns (anion exhange silca resin columns)

Plasmid Maxi Kit Test Data

Figure 1. Plasmid DNA was extracted using the GeneaidTM Maxi Plasmid Kit. The purified supercoiled Plasmid DNA [400 ml overnight E. coli (DH5α) culture, containing a 3 kb plasmid pBluescript (A600 > 2 U/ml, OD600 = 4.0)], was used in EcoRI digestion and analyzed by electrophoresis on a 1% agarose gel. M = Geneaid 1 Kb DNA Ladder

M 1 2

Test DNA Conc. 260/280 260/230 Yield 1 1550.1 µg/ml 1.87 2.25 1.55 mg

2 1540.1 µg/ml 1.87 2.29 1.54 mg







Introduction (PM010, PM025)The GeneaidTM Maxi Plasmid Kit was designed for plasmid DNA purification using pre-packed anion-exchange resin maxiprep columns to purify plasmid DNA from 200-1600 ml of cultured bacterial cells. TrueBlue Lysis Buffer (an optional color indicator) is included with the kit to prevent common handling errors, while ensuring efficient cell lysis and neutralization. A modified alkaline lysis method and RNase treatment are used to obtain clear cell lysate with minimal genomic DNA/RNA contaminants. Using an efficient gravity-flow procedure, plasmid DNA in the crude lysate is bound to the column and the contaminants are removed with PW Buffer. The purified plasmid DNA is eluted by a high salt buffer then precipitated with isopropanol for desalting. The entire procedure can be completed in 2.5 hours without ultracentrifuges, HPLC or other toxic reagents and the purified plasmid DNA is suitable for Transfection, Sequencing Reactions, Ligation, PCR, In-vitro Transcription, Microinjection, Restriction Enzyme Digestion and Gene Gun.


GeneaidTM Maxi Plasmid Kit EF Protocol Procedure

Geneaid TM Maxi Plasmid K it Endotoxin Free

Advantages• High Yield: 1.5 mg of pure transfection grade plasmid DNA from 400 ml of cultured bacterial cells• Efficient endotoxin removal (<0.1 EU/µg DNA) verified by LAL • 200-1600 ml of cultured bacterial cells• Elution Volume: 500 µl ~ 2 ml• Storage: dry at room temperature (15-25ºC)


Components• PM1, PM2, PM3 Buffers• TrueBlue Lysis Buffer• PEQ Buffer• PW Buffer• PEL Buffer• PER Buffer• RNase A• Plasmid Maxi Columns (anion exhange silica resin columns)

Plasmid Maxi Kit EF Test Data

Figure 2. The GeneaidTM Maxi Plasmid Kit Endotoxin Free includes the Plasmid Maxi Column designed to ensure high plasmid DNA yields from a wide variety of bacterial strains.







Figure 1. Plasmid DNA was extracted using the GeneaidTM Maxi Plasmid Kit. The purified supercoiled Plasmid DNA [400 ml overnight E. coli (DH5α) culture, containing a 3 kb plasmid pBluescript (A600 > 2 U/ml, OD600 = 4.0)], was used in EcoRI digestion and analyzed by electrophoresis on a 1% agarose gel. M = Geneaid 1 Kb DNA Ladder

M 1 2

Test DNA Conc. 260/280 260/230 Yield 1 1550.1 µg/ml 1.87 2.25 1.55 mg

2 1540.1 µg/ml 1.87 2.29 1.54 mg

Introduction (PME10, PME25)The GeneaidTM Maxi Plasmid Kit Endotoxin Free was designed for plasmid DNA purification using pre-packed anion-exchange resin columns to purify plasmid DNA from 200-1600 ml of cultured bacterial cells. TrueBlue Lysis Buffer (an optional color indicator) is included with the kit to prevent common handling errors, while ensuring efficient cell lysis and neutralization. PER Buffer is included with the kit to ensure efficient endotoxin removal from plasmid preparations. A modified alkaline lysis method and RNase treatment are used to obtain clear cell lysate with minimal gDNA and RNA contaminants. Using an efficient gravity-flow procedure, plasmid DNA in the crude lysate is bound to the column and the contaminants are removed with PW Buffer. The purified plasmid DNA is eluted by a high salt buffer then precipitated with isopropanol for desalting. The entire procedure can be completed in 2.5 hours without ultracentrifuges, HPLC or other toxic reagents and the purified plasmid DNA is suitable for Transfection, Sequencing Reactions, Ligation, PCR, In-vitro Transcription, Microinjection, Restriction Enzyme Digestion and Gene Gun.


Advantages• Purify 96 wells of plasmid DNA in 25 minutes!• Sample volume: 1-5 ml of cultured bacterial cells• Yield: 4-6 μg/ml of E.coli culture (high-copy)• Binding Capacity: 35 μg per well• Elution Volume: 60 μl from 100 μl elution buffer volume• Centrifuge or vacuum manifold protocol• Storage: dry at room temperature (15-25ºC)

ApplicationsFluorescent and Radioactive Sequencing, Restriction Enzyme Digestion, Library Screening, Ligation, Transformation and PCR

Components• P1, P2, P3 Buffers• Wash Buffer• Elution Buffer (10 mM Tris-HCl, pH8.5 at 25°C)• RNase A• PrestoTM Plasmid 96 Well Filter Plates• PrestoTM Plasmid 96 Well Binding Plates• 96 Deep Well Plates and 0.35 ml Collection Plates• Adhesive Film

PrestoTM 96 Well Plasmid Kit Protocol Procedure

PrestoTM 96 Well Plasmid Kit Sequencing Data

Figure 1. Sequencing data of (pBluescript) plasmid DNA purified using the PrestoTM 96 Well Plasmid Kit.

Presto TM 96 Wel l Plasmid K it

Harvest cultured bacterial cells followed by resuspension, lysis and neutralization

Cell debris filtration using the PrestoTM Plasmid 96 Well Filter Plate

Elution of pure plasmid DNA into the 0.35 ml Collection Plate

Elution of pure plasmid DNA into the 0.35 ml Collection Plate

DNA Binding then washing of contaminants

Washing of contaminants

DNA Binding

Introduction (96PDV04/10, 96PDC04/10)The Presto™ 96 Well Plasmid Kit was designed for high-throughput plasmid DNA purification from 1-5 ml of cultured bacterial cells. A modified alkaline lysis method and RNase treatment are used to obtain clear cell lysate with minimal genomic DNA/RNA contaminants. In the presence of chaotropic salt, plasmid DNA in the lysate binds to the glass fiber matrix of each well of the Presto™ Plasmid 96 Well Binding Plate. Contaminants are removed with a Wash Buffer (containing ethanol) and the purified plasmid DNA is eluted by a low salt Elution Buffer, TE or water. DNA phenol extraction or alcohol precipitation is not required and the entire procedure can be completed within 25 minutes.


Advantages• High Efficiency: up to 35 µg of pure plasmid DNA per well• Consistent high through-put recovery• The new column tip design ensures uninhibited flow-through while eliminating cross contamination of adjacent wells• 1-5 ml of bacterial culture per well• Glass fiber membrane optimized for plasmid DNA purification• Red o-ring stabilizes the glass fiber membrane during high-speed centrifugation and vacuum• Storage: dry at room temperature (15-25ºC) • Cost effective

CompatibilityGeneaid Vacuum Manifold, QIAvac 96 vacuum manifold and other vacuum manifolds, 96 well automated workstations, multi-channel pipettors, 96 Well plate centrifugation systems

Presto™ Plasmid 96 Well Filter Plate

Introduction (96PFP01)The Presto™ Plasmid 96 Well Filter Plate provides high-throughput filtration of bacterial cell debris. The yellow o-ring stabilizes the white filter membrane during vacuum filtration and high-speed centrifugation and the column tip ensures uninhibited flow-through of plasmid DNA while eliminating cross contamination of adjacent wells. Presto™ Plasmid 96 Well Filter Plates are the high performance and cost effective solution for large scale cell debris

Advantages• Consistent high through-put filtration for use with plasmid DNA purification kits to effectively remove cultured bacterial cell debris• The new column tip design ensures uninhibited flow-through while eliminating cross contamination of adjacent wells• Pink o-ring stabilizes the filter membrane during high-speed centrifugation and vacuum• Storage: dry at room temperature (15-25ºC) • Cost effective

CompatibilityGeneaid Vacuum Manifold, QIAvac 96 vacuum manifold and other vacuum manifolds, 96 well automated workstations, multi-channel pipettors, 96 Well plate centrifugation systems

Presto TM Plasmid 96 Wel l B inding Plate

Introduction (96PBP01)The Presto™ Plasmid 96 Well Binding Plate provides high-throughput purification of plasmid DNA from 1-5 ml bacterial cultures. The glass fiber membrane is optimized for binding plasmid DNA. The red O-ring stabilizes the white membrane during high-speed centrifugation and the column tip ensures uninhibited flow-through while eliminating the risk of cross contamination of adjacent wells. With plasmid DNA yields of up to 35 µg from a broad plasmid size range (1-20 kb), Presto™ Plasmid 96 Well Binding Plates are the high performance and cost effective solution for large scale plasmid DNA purification.

96 Well Column Speci�cations

1. Red O-ring stabilizes the membrane during high-speed centrifugation2. White membrane designed specifically for plasmid DNA binding 3. Column tip ensures uninhibited flow-through while eliminating cross contamination of adjacent wells

96 Well Column Speci�cations

1. Pink O-ring stabilizes the membrane during high-speed centrifugation2. White membrane designed to effectively remove cultured bacterial cell debris3. Column tip ensures uninhibited flow-through while eliminating cross contamination of adjacent wells

plasmid dna puri˜cation - geneaid · plasmid dna purification page 2 figure 2. geneaid pdh columns...

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