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AuPreP™ Blood Genomic DNA Maxi
Cat. #: GBX51-192LT
AuPreP™ Blood Genomic DNA Maxi provides a simple and fast way to purify genomic DNA (including viral or mitochondrial DNA) from various sources such as blood, plasma, serum, buffy coat, lymphocytes and body fluids. A simple spin column procedure can purify pure DNA (approximately 20-30 kb fragment) for PCR, enzymatic reactions, and other downstream application. 5 ml whole blood volume will yield 100- 300µg of genomic DNA.

Kit Contents:
 
EX Buffer

65 ml
WS Buffer 45 ml * 2
Proteinase K powder 15 mg
Blood Genomic DNA Maxi Column 10
50 ml Collection Tube 10
Protocol 1
 

Notes:
  • All procedures should be done at room temperature.
  • Prepare a 60°C (and an optional 70°C) water bath.
  • Add 180 ml of 98% ethanol to each WS bottle when first open.
  • RNA may also co-purify with genomic DNA, co-purified RNA will not inhibit PCR reaction, but may inhibit some downstream enzymatic reactions. If RNA-free genomic DNA is required, add 250 µl of 50 mg/ml RNase A (DNase free) to the sample.

Protocol:
 
  • Pipette 5 ml sample into a 50 ml tube.
    Samples: Whole blood, plasma, serum, buffy coat, body fluids, or 108-109 lymphocytes in 5 ml PBS.
    If the sample volume is less than 5 ml, add the appropriate volume of PBS to make up 5 ml.

  • Add 600µl ddH2O to the Proteinase K powder tube (provided) and vortex for 1 minute to completely dissolve Proteinase K.
    The completely dissolved Proteinase K should look transparent, if the tube looks turbid, keep vortex until complete resolution of Proteinase K. The concentration of dissolved Proteinase K is 25 mg/ml. Store the rest of Proteinase K solution at 4℃.

  • Add 50µl Proteinase K and 5 ml EX Buffer to the sample. Mix immediately by vortexing for 20 seconds.
    If sample volume is larger than 5 ml, increase the amount of EX Buffer and Proteinase K proportionally. Do not add Proteinase K directly to EX Buffer.

  • Incubate at 60°C for 30 minutes to lyse the sample, then turn the incubator to 70°C and incubate 30 minutes. Vortex or invert mix the sample every 3-5 minutes during incubation.
    Alternatively, place the sample to another 70°C incubator and incubate for 20 minutes. Sample after complete lysis should not contain insoluble residues and appear viscous.

  • Preheat ddH2O or 10 mM Tris-HCl, pH 9.0 to 70°C (5 ml /prep) for DNA elution.

  • Add 5.5 ml of isopropanol or ethanol (96-100%) to the 70°C incubated sample of step 4 and mix by vortexing.
    If the sample volume is larger than 5 ml, increase the amount of isopropanol or ethanol proportionally.

  • Place a Genomic DNA Maxi Column in a 50 ml Collection Tube (provided). Apply all the mixture from step 6 to the Genomic DNA Maxi Column, and centrifuge at 2,500 x g (3,000 rpm for a swing-bucket centrifuge) for 3 minutes. Decant the filtrate in the 50 ml tube, and place the Genomic DNA Maxi Column back to the tube.
    If a precipitate formed from step 6, apply the precipitate and mixture to the Genomic DNA Maxi Column. If Genomic DNA Maxi Column clogged after 3 minutes spin, centrifuge again at full speed for another 3 minutes.

  • Add 12.5 ml of WS Buffer. Centrifuge at 2,500 x g (3,000 rpm for a swing-bucket centrifuge) for 3 minutes, and discard the filtrate.
    Add 180 ml of ethanol (96-100%) when first open the WS Buffer bottle.

  • Add another 12.5 ml of WS Buffer. Centrifuge at 2,500 x g (3,000 rpm for a swing-bucket centrifuge) for 3 minutes, discard the filtrate, and at full speed (about 4,000 rpm) for a further 5 minutes to dry the column.

  • Place the Genomic DNA Maxi Column in a new 50 ml tube (provided by user), and discard the Collection tube contains the filtrate.

  • Elute the DNA with 5 ml of preheated ddH2O or 10 mM Tris-HCl, pH 9.0 from step 5. Centrifuge at 2,500 x g (3,000 rpm for a swing-bucket centrifuge) for 10 minutes.
    Incubate the 5 ml ddH2O or TE loaded column-tube 5 minutes at 70oC will increase DNA yield.

  • Store eluted DNA at -20°C.
    EDTA in TE elution buffer may inhibit PCR reaction, use ddH2O elution for PCR is recommended.

Troubleshooting:
 

1. Brown color residues remain on the membrane of Genomic DNA Maxi Column after washing
  • Incomplete digestion of Hemoglobin by Proteinase K.
    Prepare a new sample, add 100 µl (double amount) Proteinase K stock (25 mg/ml) to 5 ml EX Buffer and vortex thoroughly, and then incubate for 1 hour at 60oC to completely digest Hemoglobin.
  • No alcohol added to the sample before loading onto the Genomic DNA Maxi Column.
    Repeat the procedure with a new sample.
  • Incorrect amount of ethanol added to the WS Buffer.

2. Little or no DNA in the elute
  • Too low concentration of sample used.
    Increase the sample volume and repeatedly load into the Genomic DNA Maxi Column.
  • Incomplete cell lysis due to insufficient mixing of the sample with EX Buffer.
    Vortex the sample thoroughly with EX Buffer.
  • No alcohol added to the sample before loading onto the Genomic DNA Maxi Column.
    Repeat the procedure with a new sample.
  • Elution buffer (ddH2O or 10 mM Tris-HCl, pH 9.0) does not be heated to 70°C.
    Repeat elution with heated ddH2O and incubate for 5 minutes at 70°C before spin.
  • The pH of Tris buffer is too low.
    The pH of 10 mM Tris-HCl must be 9.0.
  • Elute the DNA with less than 5 ml of elution buffer.
    Less than 5 ml of elution buffer will reduce yield.

3. A260/280 ratio for genomic DNA is low
  • Inefficient cell lysis.
    Thoroughly vortex the mixture of sample.
  • Inefficient protein degradation.
    After adding Proteinase K, extend the 60°C incubation time.
  • No alcohol added to the sample before loading onto the Genomic DNA Maxi Column.
    Repeat the procedure with a new sample.
  • Incorrect amount of ethanol added to the WS Buffer.

4. A260/280 ratio for genomic DNA is high (over 1.9)
  • a. RNA contamination.
    Use RNase A in step 3 of the protocol.
 
 
 
Add RNase A into VP1 Buffer before use and store at 4°C (refer to Important Notes, No. 3, page 7). The final concentration of RNase A in VP1 Buffer is 100 µg/ml.

Volumes of buffers provided in PMD12-143LT and PMD12-145LT are enough for regular preparation of high copy plasmids from a 25-50 ml culture. Where there is a requirement of additional buffers for preparation of low copy plasmids from a larger volume of culture or for other applications, separate order for extra buffers and RNase A will be required according to user's need.

Buffers and RNase A are available for separate purchase.
Shipping and Storage
All components of AuPrePTM Plasmid Midi Kit are stable at 20-25°C for one year. If room temperature is always above 25°C, RNase A solution is better be stored at 4°C.

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.

  • Briefly centrifuge RNase A tube to bring down the solution. Add 1 ml of VP1 Buffer into RNase A solution and mix well. Transfer the mixture into VP1 Buffer bottle and store at 4°C.

  • If precipitation forms in VP2 Buffer, incubate at 55°C for 10 minutes to redissolve the salt precipitates. Do not shake VP2 Buffer, SDS present will lead to serious foaming.

  • Pre-chill VP3 Buffer on ice or at 4°C before use.

  • Prepare a 4°C centrifuge before starting the procedure. 6. Prepare room-temperature isopropanol and 70% ethanol before starting the procedure.

Protocol:
 
1. Inoculate 1-3 ml LB medium containing appropriate antibiotic(s) with plasmidcontaining bacteria from a single colony on a fresh plate or a glycerol stock. Incubate this starter culture at 37°C for 8- 16 hours with vigorous agitation.

 
2. Dilute the starter culture by 500 folds in 25-50 ml (for high copy plasmid) or 50- 100 ml (for low copy plasmid) LB medium containing appropriate antibiotic(s). Incubate at 37°C 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.

If use more than 50 ml culture for plasmid extraction, add double volumes of VP1, VP2, and VP3 Buffer to ensure complete cell lysis.

To increase yield of low copy plasmid, refer to AuPrePTM's Hints, No. 2, page 16.
3. Harvest the cells by centrifuging at 6,000 x g for 15 minutes. Decant the supernatant and remove all medium residue by pipet. Make sure that cells are well-pelleted at the bottom of the centrifuge bottle.
4. Add 4 ml of VP1 Buffer to the pellet, and resuspend the cells completely by vortexing or pipetting. Make sure that RNase A has been added into VP1 Buffer when first open (refer to Important Notes, No. 2, page 7).

No cell clump should be visible after resuspension of the cells. Clumped cells cannot be lysed well.
5. Add 4 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 and leads to chromosomal DNA contamination. If necessary, inverting and rotating the tube until the lysate becomes clear and viscous.

Do not incubate in VP2 Buffer for more than 5 minutes.
6. Add 4 ml ice-cold VP3 Buffer, mix the solution immediately and gently. A white precipiate should be formed. Pre-chill VP3 Buffer before use.

Addition of VP3 without immediate mixing will result in uneven precipitation.
7. (Optional) Incubate on ice for 20 minutes. Incubation on ice is optional. This step facilitates more complete precipitation of cell debris, proteins, chromosomal DNA, and detergent for subsequent removal by centrifugation.
8. Equilibrate a Midi-V100 Column by applying 10 ml of VP4 Buffer and allow the buffer to flow through the column by gravity. Discard the flow-through.  
9. Centrifuge the precipitate lysate from Step 6 or 7 at 20,000 x g for 30 minutes at 4°C. 20,000 x g is equivalent to 12,000 rpm in Beckman JA-17 rotor and 13,000 rpm in Sorvall SS-34 rotor.

A compact white pellet should be formed after centrifugation.
10. After centrifugation, immediately load the supernatant into the column. Allow it to flow through by gravity. Discard the flow-through. Be careful not to transfer any white pellet into the column to avoid clogging of the column. If some pellet accidentally gets into the column, use a clean sterile pipette tip to take as much out as possible.
11. Wash the column by adding 15 ml of VP5 Buffer into the column and allow it to flow through by gravity. Discard the flow-through.  
12. Add 5 ml VP6 Buffer into the column to elute plasmid DNA by gravity flow.  
13. Precipitate plasmid DNA by adding 3.75 ml (0.75 volume) of room-temperature isopropanol to the DNA eluant. Mix and centrifuge at µ 15,000 x g for 30 minutes at 4°C. Remove the supernatant carefully. 15,000 x g is equivalent to 9,500 rpm in Beckman JA-17 rotor and 11,000 rpm in Sorvall SS-34 rotor.

A transparent layer of plasmid DNA pellet formed after centrifugation can be hardly visible. Be careful NOT to pour it off together with the supernatant.
14. Wash the DNA pellet with 5 ml of roomtemperature 70% ethanol and centrifuge at µ15,000 x g for 10 minutes. Remove the supernatant carefully. Room-temperature 70% ethanol removes salts more completely than ice-cold 70% ethanol.
15. Allow the DNA pellet to air dry for 10 minutes. Redissolve the DNA in 100µl or a suitable volume of ddH2O, TE buffer, or 10 mM Tris-HCl (pH 7-8.5). For long-term storage, TE buffer should be used for elution. However, since EDTA in TE buffer may affect further enzymatic reaction, ddH2O or 10 mM Tris-HC is preferred.
16. Load the DNA solution into a Filter Column sitting in a 2-ml sterile eppendorf tube. Centrifuge the column at full speed in a microcentrifuge for 20 seconds. Collect the flow-through plasmid solution. This step removes any resin residue possibly eluted out with the plasmid.
17. Store plasmid DNA at 4°C or -20°C.  
 

Troubleshooting:
 
Problem Possible Reason Solution
Poor bacterial growth

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

 Always inoculate a big volume of LB medium with a fresh starter culture as indicated in the protocol.
  Incubation with inadequate shaking Grow cells with vigorous shaking (e.g. 250-300 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 50 ml culture for high copy plasmid.

Up to100 ml culture for low copy plasmid.

When a culture is more than 50 ml, use double volumes of VP1, VP2, and VP3 Buffer to ensure complete cell lysis.
  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.
 
 

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