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AuPreP™ DNA easy Plant Mini Kit
Cat. # PLM69-104LT(50 preps/kit)
PLM69-106LT(250 preps/kit)
AuPreP™ DNA easy Plant Mini Kit is specially designed for rapid isolation of genomic DNA (including virus, chloroplast or mitochondria) from a wide variety of plant and fungal species. The system provides shearing tubes for simple and fast homogenization as well as filtration of tissues. The simple spin-column method can isolate genomic DNA of predominantly 20-30 kb free of protein and salt contaminants without need of performing time-consuming phenol/chloroform extraction and ethanol precipitation.
Sample Plant or fungal tissues
Maximum Amount 100 mg*
DNA Yield Up to 40µg**
Preparation time 30 minutes + sample grinding time***
 

Downstream Applications
  • PCR
  • Restriction Analysis
  • Southern Blotting

Shipping and Storage
All components of DNA easy Plant Mini Kit including lyophilized Rnase A are stable at room temperature (20-25°C) for one year.
 

Kit Contents:
  PLM69-104LT (50 preps) PLM69-106LT (250 preps)
PX1 Buffer

24 ml

 120 ml
PX2 Buffer 8 ml 40 ml
PX3 Buffer 18 ml 90 ml
WS Buffer 15 ml* 45 ml x 2 **
Rnase A 20 mg 110 mg***
Plant Genomic DNA Mini Column 50 pieces 250 pieces
Collection Tube 100 pieces 500 pieces
Shearing Tube (For Mini column) 50 pieces 250 pieces
Protocol 1 1
 
  • For PLM69-104LT (50 preps), add 20 ml of 98-100% ethanol into each WS Buffer bottle when first open.
  • For PLM69-106LT (250 preps), add 180 ml of 98-100% ethanol into each WS Buffer bottle when first open
  • Add 0.2 ml sterile ddH2O to reconstitute 20 mg Rnase A or 1.1ml sterile ddH2O to reconstitute 110mg Rnase A. Vortex until Rnase A has been completely dissolved. The concentration of the stock solution is 100mg/ml. Store the solution at 4°C.

Notes:
Please read the following notes before starting the procedures
  • PX1, PX2, and PX3 Buffer contain irritants. Appropriate safety apparels such as gloves and lab coat should be worn to protect from skin contact.
  • Prepare a 65°C water bath or incubator.
  • If precipitate occurs in PX1 or PX3 Buffer, warm at 65°C to redissolve. Do not keep PX1 and PX3 Buffer at 65°C for a long time.
  • Add 0.2 ml sterile ddH2O to reconstitute 20 mg RNase A (for PLM69-104LT) or 1.1 ml sterile ddH2O to reconstitute 110 mg (for PLM69-106LT) RNase A. Vortex until RNase A has been completely dissolved. The concentration of the solution is 100 mg/ml. Store the solution at 4°C.
  • Do not add and keep RNase A directly in PX1 Buffer.
  • Add ethanol (98-100%) into WS Buffer bottle when first open. For PLM69-104LT (50 preps), add 20 ml ethanol into each WS Buffer bottle. For PLM69-106LT (250 preps), add 180 ml ethanol into each WS Buffer bottle. Ethanol is provided by the user.

Protocol:
Please read the following notes before starting the procedure
  • Do not use more than the suggested maximum amount of sample. For some samples, even are used at lower than the maximum amount, may still be difficult to be lysed completely. Thus, you are advised to start with half of the maximum amount of sample (e.g. 50 mg tissue). When you find no problem in obtaining a completely lysed sample, you would increase the sample amount in next preparations.
  • Expect for the centrifuge steps are done at the speed indicated in the protocols, all other centrifuge steps are done at full speed (10,000 x g or 13,000-14,000 rpm) in a microcentrifuge.
  • After each vortexing step, briefly centrifuge the tube to bring down the sample attached inside the cap to avoid generation of aerosols and contact with sample when the tube is opened.
  • When sample or buffer is added into the column, avoid touching the rim.
  • DNA can be eluted in Buffer TG, Milli-Q, double-distilled H2O, 10 mM Tris-HCl, or TE buffer. Since DNA elution takes place most effectively in pH 9, using H2O, (10 mM Tris-HCl,pH 9.0) or TE buffer of this pH can ensure optimal DNA elution.
1. Grind up to 100 mg plant sample under liquid nitrogen to a fine powder and quickly transfer to a sterile 1.5-ml or 2-ml eppendorf tube..

Completely ground sample gives the maximum yield of DNA. Do NOT allow the sample to thaw.
2. Add 400µl PX1 Buffer and 4 µl RNase A stock solution (100 mg/ml) to the tissue powder and vortex vigorously. Incubate the mixture at 65°C for 10 minutes. Invert mix every 2 minutes during incubation. Reconstitute RNase A powder with 200 µl dd H2O and store at 4 °C. Do NOT and keep RNase A directly in PX1 Buffer.
3. Meanwhile, preheat dd H2O (pH 9.0), 10 mM Tris- HCl (pH 9.0), or TE buffer (500 µl/prep) at 65°C for DNA elution.  
4. Add 130µl PX2 Buffer to the lysate, and vortex the mixture. Incubate it on ice for 5 minutes. If the lysate becomes very viscous after incubation on ice, centrifuge the lysate for 5 minutes and use only the supernatant.
5. Place a Shearing tube onto a Collection tube. Apply lysate (or lysate supernatant) to the Shearing tube and centrifuge for 2 minutes. Transfer the flow-through lysate from the Collection tube to a new sterile tube. Avoid pipetting any debris and pellet at the bottom of the collection tube.
6. Determine the volume of flow-through lysate obtained. Add 0.5 volume of PX3 Buffer and mix by pipetting. Add 1 volume of 98-100% ethanol to the mixture and mix by pipetting. (E.g., for 450µl lysate, add 225 µl PX3 Buffer and 450 µl ethanol) Do NOT add and keep ethanol directly in PX3 Buffer.

Do NOT centrifuge the sample after ethanol is added.
7. Place a DNA easy Plant Mini column onto a Collection tube. Apply 650 µl of the ethanol-added sample (including any precipitate) from step 6 to the column, close the cap, centrifuge at 8,000 x g (10,000 rpm) for 1 minute. Discard the flow-through. If sample remains in the column, centrifuge for 1 minute again at full speed.
8. Repeat step 6 for the rest of the sample.  
9. Wash the column twice with 0.7 ml WS Buffer by centrifuging for 30-60 seconds. Discard the flowthrough. Make sure that ethanol has been added into WS Buffer bottle when first open.
10. Centrifuge for another 2 minutes to remove any ethanol residue. Residual ethanol can affect the quality of DNA and inhibit subsequent enzymatic reactions. If necessary, centrifuge the column for a few minutes more to remove all ethanol residues.
11. Transfer the column onto a new 1.5-ml tube. Discard the collection tube and flow-through.  
12. Elute DNA with 200 µl (or 100 µl x 2) (or at least 50 µl) elution buffer such as dd H2O (pH 9), 10 mM Tris- HCl (pH 9.0), or TE buffer preheated at 65°C. Stand for 5 minutes, and centrifuge for 1-2 minutes. Elute DNA according to the yield expected; use 50 µl for less than 1 µg DNA and 200 µl x 2 for more than 20 µg DNA. Eluting DNA twice, e.g. with 100 µl elution buffer, yields more DNA in total than once with 200 µl elution buffer. Eluted DNA can be collected in the same or separate tubes.
13. Store eluted DNA at 4°C for frequent use or -20°C for long-term storage. For long-term storage, TE buffer should be used for elution. However, since EDTA in TE may affect further enzymatic reaction, H2O or 10 mM Tris-HCl are preferred for elution of DNA immediately used for further enzymatic reactions.
 

Troubleshooting Guide:
 
Problem

Possible Reason

 Solution
Low or no yield of DNA Insufficient grinding of the sample Complete sample grinding ensures maximum extraction of DNA
  Sample is from old tissues or contains very low number of cells Use young tissues or those, e.g. epidermis, that are expected to contain higher percentage of cells
  Sample is not lysed completely due to too much is used Start with half of the suggested amount.
  Sample is not lysed completely due to insufficient mixing Invert mix the sample every 2 minutes during incubation
  Incorrect amount of PX3 Buffer or ethanol is added to the sample before loaded into the column Determine the volume of throughflow lysate. Add 0.5 volume of PX3 Buffer and 1 volume of 98-100% ethanol.
  WS Buffer does not contain ethanol. Make sure that ethanol is added into the WS Buffer bottle when first open.
Low or no yield of DNA Elution solution (Buffer TG, ddH2O of pH 8-9, or TE) is not preheated at 65°C. Preheat elution buffer at 65°C before used.
  pH of the elution buffer is too low Make sure that the pH of elution buffer is 9.
  Use 200 µl elution buffer Use as little as 50µl elution buffer to elute DNA (<1 µg) when low amount of sample is applied.
Column is clogged when passing the sample Sample is too viscous Too much sample is used. Reduce the sample amount.
    Sample contains a lot of sticky secondary metabolites. Use young tissues or those, e.g. epidermis, that contains higher percentage of cells but lower amount of secondary metabolites.
  Lysate contains insoluble materials Avoid pipetting any debris and pellet at the bottom of the collection tube after passing Shearing tube.
  Insufficient centrifugation Centrifuge the column at full speed
DNA of poor quality Eluted genomic DNA contains ethanol residue After the second wash, discard the flow-through, centrifuge the column at full speed for another 2 minutes to remove the ethanol residue completely
A260/A280 ratio of eluted genomic DNA is high (>1.9) Eluted genomic DNA contains a lot of RNA Add Rnase A into the sample as described in the protocol. Do NOT add and keep RNase A directly in PX1 Buffer.
Genomic DNA appeared smearing and degraded Sample is not fresh or stored improperly for long time Flash freeze fresh sample under liquid nitrogen and store at -80°C if not used immediately.
  Sample loaded into Shearing tube is too viscous Remove precipitates from the viscous sample by centrifuging at full speed for 5 minutes and load the supernatant into the Shearing tube.
 

AuPrePTM DNA easy Plant Mini Kit

AuPrePTMs Hints:
  • Low yield or purity of genomic DNA is usually due to incomplete lysis of the sample. Starting with a maximum amount of sample does NOT usually give the best yield of DNA. Instead, it always results into incomplete sample lysis and denaturation of proteins, thus making extracting of all DNA from the sample unfeasible. Further it always requires subsequent removal of undigested residues and yields viscous sample lysate. When the lysate is too viscous, it may have difficulty to pass through the column easily, and more importantly, it usually indicates the presence of an abundant amount of contaminants such as proteins and salts. Contaminants of high amount not only affect DNA binding, but also may not be washed off completely, thus leading to carry over to the eluted genomic DNA. Therefore, a good quality and yield of DNA is only expected when a sample is completely lysed. We recommend starting with half of the maximum amount of sample suggested. When there is no problem in sample digestion and passing lysate through the column, amount of sample to be applied can be increased gradually in the subsequent preparations.
  • Young plant tissues are preferred because they give higher yield and better quality of genomic DNA due to that they contain a higher percentage of cells but lower amount of polysaccharides and secondary metabolites, which always make sample handling uneasy and interfere with OD readings if eluted with DNA. For dried sample, using an amount 2-3 folds lower than that for fresh one is advised because dried sample contain more cells per weight; using the product’s suggested sample amount for dried sample will lead to inefficient lysis.
  • DNA should not be eluted in ddH2O for storage because it is degraded gradually through acid hydrolysis. Since DNA is more stable in a slightly alkaline (pH 7.5-9) buffering condition, 10 mM Tris- HCl is considered a better choice than water.
  • Use ddH2O of acidic pH (5.0-6.0) to dilute DNA and RNA samples for spectrophotometric analysis will significantly decrease A260/A280 ratio of the sample (Wilfinger et al., 1997). ddH2O of pH 7-8 or 10 mM Tris-HCl of pH 8-8.5 should be used to dilute the samples.
  • Higher DNA recovery can be attained by eluting the column twice. That is, eluting twice with eg, 100µl ddH2O or buffer, yeilds more DNA in total than eluting once with 200µl ddH2O or buffer.
 
 

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