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AuPreP™ Plasmid Maxi Kit
Cat No. PMX12-162LT
Cat No. PMX12-163LT
Description
AuPrePTM Plasmid Maxi Kit allows the isolation of ultrapure plasmid DNA from up to 500 ml culture. Plasmid DNA purified from AuPrePTM's proprietary anion-exchange resin, is suited for use in transfection, automated sequencing and enzymatic modification.
Parameter Value
Average preparation time

120 ~ 150 minutes
Workable length of fragment 1.5-kbp ~ 150-kbp
Maximal recovery 99 %
Minimal elution volume 10 ml
Maximal capacity >500μg
Regular sample volume 100 ml ~ 500 ml
 

Downstream Applications
  • Restrictive enzymatic digestion
  • Modifying enzymatic reaction
  • Sequencing & PCR * Ligation
  • Labeling and Hybridization
  • Radioactive and Fluorescent sequencing
Product Contents
 
  PMX12-162LT (25 preps) PMX12-163LT (50 preps)
VP1 Buffer

120ml

 265ml
VP2 Buffer 120 ml 265 ml
VP3 Buffer 120 ml 265 ml
VPN Buffer 225 ml x 2 265 ml x 4
VPE Buffer 120 ml 265 ml
Plasmid Maxi Column 10 pieces 25 pieces
Filter Column 20 pieces 50 pieces
RNase A, (20 mg/ml) 0.600 ml 1.325 ml
Protocol 1 1
 

Buffers are available for separate purchase. Please contact us for ordering information.
 
Shipping and Storage
All components of AuPrePTM Plasmid Maxi Kit are stable at 20-25°C for one year. Product should be stored in a dry place and kept away from direct sunlight. If room temperature is always above 25°C, RNase A solution is better be stored at 4°C. Product should be stored in a dry place and kept away from direct sunlight.
Important Notes

Please read the following notes before starting the procedures.
  • Buffers provided in this system contain irritants. Appropriate safety apparels such as gloves and lab coat should be worn. People handling the kit may need suitable instruction.
  • All procedures should be done at room temperature (20-25ºC) and centrifugation should be done at full speed (10,000 x g or 13,000 rpm), unless otherwise notified.
  • Spin RNase A solution tube before use, apply all of RNase A solution into VP1 Buffer bottle and mix well to store at 4ºC.
  • If precipitation forms in VP2 Buffer, incubate at 55ºC for 10 minutes to redissolve the salt precipitates. Do not shake VP2 Buffer; SDS present will lead to serious foaming.
  • Sit VP3 Buffer on ice before use.
  • The volume of VP-3 Buffer used in the protocol is developed for specific volume of sample culture. If starting sample culture is larger than 50/100ml, please increase the volume of VP1-3 buffer proportionally.
  • Please be aware that there are corresponding important notes listed below each step of the protocol. Important hints for user’s references are listed beside the corresponding paragraph of the protocol. This information has been provided to help users minimize any potential problem.

Protocol for PMX12-162LT/163LT:
 
1. Culture plasmid-containing bacterial cells in 100-200 ml (high-copy-number plasmids) or 300-500 ml (lowcopy- number plasmids) of LB medium. Grow 12-16 hours with vigorous shaking at 37ºC.

Bacterial cells should not be grown more than 16 hours, and over-grown cells usually results in low plasmid yield and quality. Plasmids with high quality and yield always come from good bacterial sample.
2. Harvest the bacterial cells by centrifuging at 6,000 x g for 15 minutes. Make sure that cells are well-pelleted at the bottom of the centrifuge bottle.
3. Equilibrate Plasmid Maxi Columns by applying 5 ml of 98-100% ethanol. Allow the column to empty by gravity flow and discard the filtrate. Before using column, shake the column to separate resins completely then gently knock the column to make all resins down to the bottom of the column.
4. Apply 10 ml of VPN Buffer to the Plasmid Maxi Column and allow it to flow through by gravity flow and discard the filtrate.  
5. Resuspend the cell pellet in 10 ml VP1Buffer. Important! No cell clump should be visible after resuspension. Clumped cells lead to low plasmid yield and quality. Make sure that RNase A has been added into VP1 Buffer when first open.
6. Add 10 ml of VP2 Buffer and gently mix (invert and rotate the tube or bottle) to lyse the cells until the lysate becomes clear. Incubate at room temperature for 5 minutes. Do not vortex! Vortexing shears genomic DNA contaminating plasmids and leading to serious foaming.
7. Add 10 ml ice-cold VP3 Buffer, mix gently by inverting. Addition of VP3 without immediate mixing will result in uneven precipitation.
8. Centrifuge at 20,000 x g for 15 minutes at 4ºC. A compact white pellet should be formed after centrifugation.
9. Apply the supernatant with plasmid DNA to the Plasmid Maxi Column and allow it to flow through by gravity flow and discard the filtrate.  
10. Wash the column once with 30 ml of VPN Buffer by gravity flow and discard the filtrate. If volume of bacterial sample is more than 200 ml, washing the column with another 15 ml of VPN with get plasmids with higher quality.
11. Apply 10 ml of VPE Buffer to elute DNA by gravity flow. If plasmid is larger than 50-kbp, warm up VPE Buffer at 40ºC.
12. Precipitate DNA by adding 10 ml (0.7 volume) of room-temperature isopropanol to the DNA eluant. Mix and centrifuge at 15,000 x g for 30 minutes at 4ºC. Remove the supernatant carefully.  
13. Wash the DNA pellet with 10 ml of room-temperature 70% ethanol and centrifuge at 15,000 x g for 10 minutes. Remove the supernatant carefully.  
14. Allow the DNA pellet to air dry for 10 minutes and dissolve the DNA in 250µl or selected volume of TE or ddH2O.  
15. < Optional Step > Some insoluble material may remain in the final product. To eliminate the insoluble material, load the dissolved DNA sample into a Filter Column (sitting in a 1.5 ml tube) and spin at full speed in a microcentrifuge for 20 seconds. Collect the eluted DNA sample in the 1.5 ml tube.  
16. Store DNA at -20º.  
 
Troubleshooting Guide
 
Problem Possible Reason Solution
Poor bacterial growth

Inoculate bacterial sample from an plate or old plate or culture stock stored for a long time period

 Always inoculate utilizing bacterial cells from a freshly streaked plate and grow with required antibiotic(s).
  Incubation with inadequate shaking Grow cells with vigorous shaking (e.g. 250 rpm). Adjust a suitable shaking speed according to the angular magnitude of the shaker platform.
Poor cell lysis Use too many bacterial cells harvested from a big volume of culture or an over-grown culture Up to 100 ml culture for high copy plasmid.

Up to 500 ml culture for low copy plasmid.

When a culture is more than 100 ml, use increased amount of VP1, VP2, and VP3 Buffer.
  Cell pellet is not well resuspended Do not add VP2 Buffer until cells are completely resuspended by vortexing or pipetting.
Low yield of plasmid DNA Not enough bacterial cells Ensure that bacteria have grown well (OD600>1) after overnight incubation with vigorous agitation.
  Overgrowth of bacteria Incubate bacterial culture with LB medium and do not incubate the culture for more than 16 hours.
  Plasmid does not propagate Always inoculate bacterial cells from freshly streaked plate and grow with required antibiotic(s).
  Poor cell lysis Refer to Solution section of Problem - "Poor cell lysis".
  Plasmid is larger than 50-kbp. Use VPE Buffer preheated to 40ºC.
  Inefficient or incomplete DNA elution. Use no less than 10 ml of VPE Buffer to elute.
Plasmid appears
smearing or degraded.		 Host strain is endA+ Use endA- strain.
  Overgrowth of bacteria Incubate bacterial culture with LB medium and do not incubate for more than 16 hours.
Genomic DNA
contamination in elute		 Lysate improperly prepared After VP2 Buffer added, mix gently to prevent genomic DNA shearing and do not incubate for more than 5 minutes
  DNA is not redissolve completely Make sure to wash all DNA off from the wall.
RNA contamination Not enough RNase A activity in VP1 Buffer Ensure that all RNase A is added into VP1 Buffer and stored at 4ºC. After long-term storage (about 6 months), add RNase A into VP1 Buffer to the conc. 100μg/ml and store at 4ºC.
Plasmid of poor DNA
quality		 Too much salt. Wash DNA pellet after isopropanol precipitation twice with 70% ethanol at room temperature.
  Used too many bacterial cells harvested from a large culture or an over-grown culture. Reduce the amount of sample used. Incubate bacterial culture with LB medium and do not incubate for more than 16 hours.
 
 

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