Plasmid DNA Isolation Protocol

Author: Victoria Fox

Introduction

The OPS Diagnostics Plasmid Isolation Kit has been designed to rapidly purify plasmid DNA from Escherichia coli. The isolation process couples traditional alkaline lysis and chaotropes with modern spin columns to accelerate purification of supercoiled plasmid DNA. For best yields, E. coli with plasmid should be cultured under selective pressure overnight and to a high cell density. Both Luria Bertani (LB) and Terrific Broth are suitable for culturing. The OPS Diagnostics Plasmid Isolation Kit is simple to use and requires only a microcentrifuge. The kit is also a cost-effective spin column-based kit as compared to comparable products.

Materials and Equipment

  • Culture E. coli with plasmid
  • Microcentrifuge tubes
  • Resuspension Buffer (RB)
  • RNase A
  • Lysis Solution (LS)
  • Neutralization Buffer (NB)
  • Silica Spin Columns (Product: SSC 100-01)
  • Wash Buffer I (WB I)
  • Wash Buffer II (WB II)
  • Elution Buffer (EB)
  • Microcentrifuge
  • Vortex
  • Micropipettes

Protocol

  1. Add 1-1.5 ml of cultured plasmid sample to a microcentrifuge tube, centrifuge the tube at 8,000 x g for 3 minutes to pellet the cells.
    • A 1.7 ml microcentrifuge tube can be used however, higher DNA yields have been observed using a 2.0 ml conical microcentrifuge tube.
  2. Carefully decant the liquid without disturbing the pellet. Completely resuspend the pellet by adding 250 µl of Resuspension Buffer to the microcentrifuge tube and vortex.
  3. Add 10 µl of RNase A to the microcentrifuge tube, mix and let stand for 1 minute.
  4. Add 250 µl of Lysis Buffer to the microcentrifuge tube and mix the sample by inverting the tube 4-6 times.
    • Do not let lysis continue for more than 5 minutes.
    • To avoid genomic DNA contamination, do not vortex.
  5. Add 350 µl of Neutralization Buffer to the microcentrifuge tube, mix the sample by inverting the tube 4-6 times. Centrifuge the sample at 13,000 x g for 5 minutes.
    • A white precipitate will form.
  6. Transfer up to 700 µl of the clear supernatant into a clean OPS spin column (provided). Centrifuge the spin column at 13,000 x g for 1 minute and discard the flow through.
  7. Add 500 µl of Wash Buffer I to the spin column. Centrifuge the spin column at 13,000 x g for 1 minute to allow the solution to pass through the spin column and discard the flow through.
  8. Wash the column with 700 µl of Wash Buffer II and centrifuge the column at 13,000 x g for 1 minute to pass through the wash solution. Discard the flow through.
  9. Centrifuge the column again at 13,000 x g for 1 minute to pass through residual buffer and discard the flow through.
  10. Place the spin column into a new microcentrifuge tube and add 50 µl of the Elution Buffer. Centrifuge the column at 13,000 x g for 1 minute.
    • Dispense the Elution Buffer onto the center of the membrane.
  11. Store the eluted DNA at -20°C.

Results

Awaiting results and gel picture

Troubleshooting Guide

Problem Possible Cause Solution
Low or No Yield General Low yields may be caused by several factors. To find the source of the problem, analyze samples saved from each step in the procedure.
Poor Bacterial Growth Inoculated bacterial cells from a well-established (old) plate or culture stock. Inoculate with a single colony of bacterial cells from a freshly prepared plate and grow with the proper antibiotics
Inadequate shaking during the incubation period Grow up cells with vigorous shaking for 12-16 hours
Poor Cell Lysis Harvested too many bacterial cells from a large or over-grown culture Only grow cells for 12-16 hours. Calculate culture cell density and adjust the volumes of RB, LS, and NB accordingly
Inadequate resuspension of the cell pellet Ensure cells have been completely resuspended in RB by vortex before adding Lysis Solution
Lysate prepared incorrectly Check storage conditions and age of buffers
Buffer precipitated Redissolve by warming to 37°C
Low Plasmid DNA Yields Not enough bacterial cells Ensure cells have been grown overnight under the proper conditions with vigorous shaking
Overgrown bacterial cells Do not incubate for more than 16 hours
Plasmid did not propagate The desired clone should be streaked onto a fresh plate with the correct selective agent so single colonies can be isolated. Repeat to inoculate with a single colony of an antibiotic-resistant clone.
Inadequate or incomplete DNA elution For complete DNA elution the elution solution must be in full contact with the membrane. No less than 50 µl should be used.
DNA Found in the Wash Flow Through Ethanol was omitted from WB II Restart procedure with the ethanol added to WBII
Genomic DNA Contamination Extended Lysis solution incubation or vigorous shaking after the addition of Lysis Solution Mix by gently inverting the tube 4-6 times, do not vortex to prevent shearing. Do not let lysis continue for more than 5 minutes.
Buffer NB not mixed thoroughly Once NB is added mix immediately by inverting the tube 4-6 times
Culture overgrown Overgrown cultures contain lysed cells and degraded DNA
RNA Contamination RNase A was omitted Add 10 µl of the included RNase A solution to the sample
Reduced RNase A activity due to improper storage Store the RNase A solution at - 20°C
Low DNA Quality Eluate contains salt Wash the column again with 750 µl WB II and centrifuging
Eluate contains ethanol Perform step 9 in the protocol

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