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AuPreP™ SPINm SPIN Miniprep Kit
Cat. No. SM27-104LT
Cat No. SM27-106LT
Description
AuPreP™ SPINm SPIN Miniprep Kit provides a simple, fast, and cost-effective method to purify plasmid DNA without phenol/chloroform extraction. This kit is optimized for increased plasmid DNA yield. It is based on binding of DNA to silica-based membranes in chaotropic salts. An average yield of 1 to 40 µg of plasmid DNA can be expected from 1 to 5 ml overnight bacterial culture.
Downstream Applications
  • Radioactive and Fluorescent sequencing
  • APD, RFLP * Restrictive enzymatic digestion
  • Transformation
  • Ligation
  • PCR
  • Library screening or Large-scale screening
 
Parameter Value
Average preparation time 20-30 minutes
Workable length of fragment 1.5-kbp ~ 15-kbp
Maximal recovery 95%
Minimal elution volume 30μl
Maximal capacity 40μg
Regular sample volume 1-5ml
 

Product Contents
  SM27-104LT
(50 Preps.)		 SM27-106LT
(250 Preps.)
MX 1 Buffer*

12ml

 60 ml
MX 2 Buffer 15ml 75 ml
MX 3 Buffer 20ml 100 ml
WN Buffer 6ml* 30 ml**
WS Buffer 10ml+ 45 ml++
Elution Buffer 5ml 25 ml
RNase A , 20mg/ml 0.042ml 0.210ml
AuPreP™ SPINm Mini Column 50 pieces 250 pieces
Collection Tube 50 pieces 250 pieces
Protocol 1 1
Add *24ml 98-100% ethanol into WN Buffer bottle for SM27-104LT (50 preps) and **120ml for (250 preps) when first open. Ethanol is not included, and has to be provided by users. Be sure to tighten the cap following each use after the ethanol has been added.

Add + 40ml 98-100% ethanol into WS Buffer bottle for SM27-104LT (50 preps) and + + 180ml for SM27-106LT (250 preps) when first open. Ethanol is not included, and has to be provided by users. Be sure to tighten the cap following each use after the ethanol has been added.

Shipping and Storage
All components of AuPreP™ SPINm SPIN Miniprep Kit are stable at 20-250C for one year. The kit should be stored in a dry place and kept away from direct sunlight. RNase A solution is better be stored at 40C.

Important Notes
Please read the following notes before starting the procedures
  • Buffers provided in this kit contain irritants. Appropriate safety apparels such as gloves and lab coat should be worn. People handling the kit may need suitable instructions.
  • All procedures should be done at room temperature (20-25°C) and centrifugation should be performed at 7,000 x g ~ 10,000 x g (9,000rpm ~ 13,000rpm) in a microcentrifuge, unless otherwise notified.
  • Briefly centrifuge RNase A tube to bring down the solution. Add 1 ml of MX1 Buffer into RNase A solution and mix well. Transfer the mixture into MX1 Buffer bottle and store at 4°C.
  • If precipitation forms in MX2 Buffer, incubate at 55°C for 10 minutes to redissolve the salt precipitates. Do not shake MX2 Buffer, SDS present will lead to serious foaming.
  • For long-term storage of the eluted DNA, TE buffer should be used for elution. Since EDTA in TE may affect downstream applications, Elution Buffer (provided) or ddH2O (pH 7.0-8.5) is preferred for elution of DNA immediately used for further enzymatic reactions.
  • Please be awared 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 problems.
 
AuPreP™ SPINm SPIN Miniprep Kit-------------------Protocol for Spin Method
 
  Related Notes
1Grow 1 to 5 ml plasmid-containing bacterial cells in LB medium with appropriate antibiotic(s) overnight (12-16 hours) with vigorous agitation.

If the bacterial cells are grown more than 16 hours, over-grown cells usually have reduced yield of plasmids (refer to AuPrep™ SPINm Hints, No. 1, page 14).
2. Pellet the cells by centrifuging for 1-2 minutes. Decant the supernatant and remove all medium residue by pipette. Make sure that cells are well-pelleted in the bottom.
3. Add 200 µl of MX1 Buffer to the pellet, and resuspend the cells completely by vortexing or pipetting. Make sure that RNase A has been added into MX1 Buffer when first open.
No cell clump should be visible after resuspension of the cells. Clumped cells cannot be lysed well.
4. Add 250µl of MX2 Buffer and gently mix (invert the tube 8-10 times) to lyse the cells until the lysate becomes clear. Incubate at room temperature for 2-5 minutes Do NOT vortex! Vortexing shears genomic DNA and leads to chromosomal DNA contamination. If necessary, continue inverting the tube until the lysate becomes clear and viscous.
5. Add 350µl of MX3 Buffer to neutralize the lysate, then immediately and gently mix the solution. A white precipitate should be formed. Addition of MX3 Buffer without immediate mixing will result in uneven precipitation.
6. Centrifuge at 10,000 x g (13,000rpm) for 5-10 minutes, meanwhile place a SPINm Column onto a Collection Tube. A compact white pellet should be formed after centrifugation.
7. Transfer the supernatant carefully into the column. Be careful not to transfer any white pellet into the column to avoid clogging of the membrane.
8. Centrifuge at 7,000 x g (9,000rpm) for 30-60 seconds. Discard the flow-through.  
9. Wash the column once with 0.5 ml WN Buffer by centrifuging at 7,000 x g (9,000rpm) for 30-60 seconds. Discard the flow-through.  
10. Wash the column once with 0.7 ml WS Buffer by centrifuging at 7,000 x g (9,000rpm) for 30-60 seconds. Discard the flow-through. Ensure that ethanol has been added into WS Buffer bottle when first open.
11. Centrifuge the column at 10,000 x g (13,000rpm) at full speed for another 3 minutes to remove residual ethanol. Residual ethanol can affect the quality of DNA and inhibit subsequent enzymatic reactions.
12. Place the column onto a new 1.5-ml centrifuge tube. Add 50µl of Elution Buffer (provided) onto the center of the membrane. For effective elution, make sure that the elution solution is dispensed onto the center of the membrane and is completely absorbed (refer to AuPreP™ SPINm Hints, No. 2 & 4, page 14).
13. Stand the column for 2-3 minutes, and centrifuge at 10,000 x g (13,000rpm) for 2-3 minutes to elute DNA. If the solution still retains on the surface, pulse-centrifuging the tube for 1-2 seconds can drag the solution into the membrane. Do NOT over-centrifuge as the solution will get out of the membrane easily.
14. Store plasmid DNA at 4°C or –20°C.  
 
AuPreP™ SPINm SPIN Miniprep Kit-------------------Protocol for Vacuum Method
 
  Related Notes
1. Grow 1 to 5 ml plasmid-containing bacterial cells in LB medium with appropriate antibiotic(s) overnight (12-16 hours) with vigorous agitation.

If the bacterial cells are grown more than 16 hours, over-grown cells usually have reduced yield of plasmids (refer to AuPreP™ SPINm Hints, Hints, No. 1, page 14).
2. Pellet the cells by centrifuging for 1-2 minutes. Decant the supernatant and remove all medium residue by pipette. Make sure that cells are well-pelleted in the bottom.
3. Add 200 µl of MX1 Buffer to the pellet, and resuspend the cells completely by vortexing or pipetting. Make sure that RNase A has been added into MX1 Buffer when first open.

No cell clump should be visible after resuspension of the cells. Clumped cells cannot be lysed well.
4. Add 250 µl of MX2 Buffer and gently mix (invert the tube 8-10 times) to lyse the cells until the lysate becomes clear. Incubate at room temperature for 2-5 minutes. Do NOT vortex! Vortexing shears genomic DNA and leads to chromosomal DNA contamination. The lysate should become clear and viscous. Insufficient cell-lysis leads to low plasmid yield and quality.
5. Add 350 µl of MX3 Buffer to neutralize the lysate, then immediately and gently mix the solution. A white precipitate should be formed. Addition of MX3 Buffer without immediate mixing will result in uneven precipitation.
6. Centrifuge at 10,000 x g (13,000rpm) for 5-10 minutes, meanwhile insert the tip of a SPINmColumn into the luer-lock of a vacuum manifold (e.g. Promega's Vac-man*). A compact white pellet should be formed after centrifugation.
7. Transfer the supernatant carefully into the column. Be careful not to transfer any white pellet into the column to avoid clotting of the membrane
8. Apply vacuum to draw all the liquid into the manifold.  
9. Wash the column once with 0.5 ml WN Buffer by re-applying vacuum to draw all the liquid. Ensure that ethanol has been added into WN Buffer bottle when first open.
10. Wash the column once with 0.7 ml WS Buffer by re-applying vacuum to draw all the liquid. Ensure that ethanol has been added into WS Buffer bottle when first open.
11. Place the column onto a Collection Tube. Centrifuge at 10,000 x g (13,000rpm) for another 3 minutes to remove residual ethanol. Residual ethanol can affect the quality of DNA and inhibit subsequent enzymatic reactions.
12. Place the column onto a new 1.5-ml centrifuge tube. Add 50 μl of Elution Buffer (provided) onto the center of the membrane. For effective elution, make sure that the elution solution is dispensed onto the center of the membrane and is completely absorbed (refer to AuPreP™ SPINm Hints , No. 2 & 4, page 14).
13. Stand the column for 2-3 minutes, and centrifuge at 10,000 x g (13,000rpm) for 2-3 minutes to elute DNA. If the solution still retains on the surface, pulse-centrifuging the tube for 1-2 seconds can drag the solution into the membrane. Do NOT over-centrifuge as the solution will get out of the membrane easily.
14. Store plasmid DNA at 4°C or -20°C. Standing the column for a further 5 minutes may result in increased yield of plasmid.
* Vac-man is a trademark of Promega Corporation.
 

Troubleshooting Guide
Problem Possible Reason Solution
Poor bacterial growth

Inoculate bacterial cells from a plate or a cultural stock stored for long time

 Always inoculate 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 an orbital shaker platform.
Poor cell lysis Use too many bacterial cells harvested from a large culture or an over-grown culture Up to 5 ml culture for high-copy plasmid Up to 10 ml culture for low-copy plasmid. When the culture is more than 5 ml, use double amount of MX1, MX2, and MX3 Buffer.
  Cell pellet is not well resuspended Do not add MX2 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 shaking.
  Overgrowth of bacteria Do not incubate for more than 16 hours.
  Plasmid does not propagate Always inoculate bacterial cells from a freshly streaked plate and grow with required antibiotic(s).
  Inefficient or incomplete DNA elution Efficient and complete DNA elution only takes place when elution solution is within pH 7-8.5 and is in full contact with the membrane. Make sure that no less than 30 l of solution is dispensed onto the membrane and is completely absorbed into it before centrifugation.
Low yield of plasmid DNA Poor cell lysis Refer to Solution section of Problem - "Poor cell lysis".
  Plasmid is larger than 10 kb Use elution solution preheated to 70C
Plasmid appears smearing or degraded Host strain is endA+ Use endA- strain if possible. Or wash with WF Buffer twice. Eluting DNA with TE buffer and storing at –20C can inhibit nuclease activity.
Genomic DNA contamination in eluate Lysate prepared improperly After MX2 Buffer is added, mix gently to prevent genomic DNA shearing and do not incubate for more than 5 minutes
RNA contamination No RNase A activity in MX1 Buffer Ensure that Rnase A is added into MX1 Buffer and stored at 4C
    Add RNase A into MX1 Buffer to a concentration of 50 g/ml and store at 4C.
 
Plasmid of poor quality    
1.Plasmid DNA cannot be digested well or does not deposit into the well of gel during loading

Ethanol in WS Buffer is not completely removed

 After washing with WS Buffer, do discard the flow-through and centrifuge the column at full speed for 3 minutes. If necessary, centrifugation for a few minutes more can completely remove ethanol.
2.Plasmid is denatured and migrates faster than supercoiled form during electrophoresis Incubate in MX2 Buffer for too long time After MX2 Buffer is added, do not incubate for more than 5 minutes.
 

AuPreP™ SPINm Hints
  • Good yield of plasmids is always guaranteed from a healthily-grown culture. If plasmid extraction cannot be done shortly, a wellgrown culture can be kept on ice for a while (<1 hour) without leading to reduction of yield. Never leave the culture on a bench for a long time as a well-grown culture starts to deteriorate due to lack of oxygen.
  • More concentrated plasmid solution can be obtained by eluting DNA in 30µl. However, this leads to about 40% loss of DNA due to incomplete elution. We recommend eluting in at least 50 µl to optimize recovery. Similarly, though eluting in volume more than 50 µl (e.g. 100µl) can have about 10% increase in recovery, this results in getting a more diluted plasmid solution. Each user should apply a suitable elution volume according to his/her needs.
  • Milli-Q or double-distilled H2O stored in a laboratory for a period of time usually becomes acidic due to dissolution of CO2 or other acidic vapor such as Hcl from air. Always check the pH to make sure that it is between 7.0 to 8.5 before used. Using H2O of pH less 7.0 for elution will lead to reduced yield of plasmid. Using H2O of acidic pH (pH 5.0-6.0) to dilute DNA or RNA samples for spectrophotometric analysis will also significantly decrease A260/A280 ratio of the sample (Wilfinger et al., 1997).
  • Using elution solution preheated to 70°C may increase plasmid yield by about 20%.
 
 

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