Plasmid DNA Purification Support—Troubleshooting

Here are some suggestions to try:

• Make sure the binding of the plasmid is being done at room temperature (RT). Temperature affects the pH of the binding solution. Make sure all other solutions were also warmed to RT.
• Verify that the centrifugation immediately following the neutralization step was not done at 4°C. If it was, the supernatant MUST be warmed to RT before binding on the column. We find that the DNA binds to the matrix of the columns better if the lysate is at room temperature.
• Low copy number plasmid may have been used. Check plasmid.
• Not all the medium may have been removed at the cell harvesting step, so the pH of the subsequent steps was affected.
• The cell pellet may not have been thoroughly resuspended in the resuspension step.
• Purified DNA may have been overdried after isopropanol precipitation and ethanol wash. Only air-dry the pellet.
• Pellet may have been lost during the isopropanol precipitation and ethanol wash. Be careful at this step, as the pellet tends to be slippery. It is best to pipette off alcohol solutions rather than pour them off.
• When working with BAC DNA (and therefore increasing the NaCl from 0.8 M to 0.9 M in the Wash Buffer W8), an excessive volume of Wash Buffer W8 may have been used. See the BAC prep section in the manual.
• Try elution of DNA with heated elution buffer: For plasmids less than 10 kb, no heating is required. For 10–30 kb, heating (65–70°C) is optional, and may increase elution efficiency by ~20%. For plasmids >30 kb, heating is recommended, and may increase elution efficiency by ~50%. Perform an additional elution to increase yield by up to 10%.
• If there is some insoluble material in the eluted DNA, it could be resin particles (resin fines). These are inert and can be removed by a centrifugation at 12,000 x g for 1 minute at RT.
• You can also try adding chloramphenicol to the culture at mid-log phase (around OD590 0.8–1.0).

Please check to see whether the plasmid was ethanol precipitated and washed after elution from the column. Inhibition may occur if there is too much salt and/or if the pH is too high.

To reduce RNA contamination:

1. Increase the volume of Precipitation Buffer (N3) by 10%. (This will reduce the amount of RNA binding to the resin.)
   
2. Increase the salt concentration in the Wash Buffer (W8) from 800 mM NaCl to 860 mM NaCl by adding 0.35 g NaCl per 100 ml Wash Buffer. (This will wash more of the RNA off the resin.)
   (NOTE: Each of these changes alone reduces the amount of RNA contamination significantly. Together these changes eliminate virtually all RNA).

• Make sure that the RNase A was added and thoroughly dissolved in the resuspension buffer.
• Make sure that the resuspension buffer containing RNase A was not stored for more than 1 year at 4°C.
• PureLink® HiPure Filter Midi and Maxi Kits may help reduce RNA contamination by clearing bacterial lysate more thoroughly.
• If RNA contamination persists, the RNase A concentration in the resuspension buffer can be increased. It can go as high as 400 µg/mL. This is particularly useful when isolating low-copy plasmids or other single-copy DNA forms such as BACs.
• Contaminating RNA tends to be a little more of a problem when low copy number plasmids are purified due to a large number of cells being processed.

To troubleshoot the bacterial culture:

• The columns may have been overloaded with too many cells. Verify that the culture used was grown to the proper OD value and that the volumes used were within kit specifications. A cell density of approximately 10^9 cells/mL or an absorbance at 600 nm (A600) of 1–1.5 is appropriate. Verify growth times: 16 to 24 hours at 37°C should be sufficient to reach desired OD. Verify the medium used, as TB or very rich broth could produce a very dense culture, which may cause reduced lysis.
• Verify volumes of cells and buffers added (resuspension with RNAse A, lysis, neutralization steps). For >200 mL culture, increased buffer volumes should be used. Check on amount of RNase added to resuspension buffer. For cultures with high density or high volumes, you could add extra RNase A, to 400 µg/mL as mentioned above.

If contaminating DNA is seen, there may have been problems with the cell lysis and neutralization steps. Perhaps the mixture was not centrifuged appropriately or the sample was mixed too harshly during the lysis step.

This is common if the solution gets too cold. If necessary, warm the solution briefly to 37°C until the precipitate is dissolved.

Please see our suggestions for this problem:

• Make sure that the column was equilibrated with the kit's Equilibration Buffer.
• Was the insoluble material completely pelleted after neutralization? This could clog the column.
• Was the supernatant after centrifuging the solution after neutralization pipetted onto the column and not just poured onto the column? It is best to pipette it onto the column.

We have seen this on occasion. The particles do not affect quality of the DNA. Remove the particles by performimg a 1 minute centrifugation at 12,000 x g.

Extra bands can occur when plasmid DNA is nicked and/or permanently denatured. Plasmid DNA that has been nicked (covalently opened) will run slower than supercoiled DNA during electrophoresis. A small amount of this species of DNA is common and is suitable for downstream applications. Permanently denatured DNA will migrate ahead of the supercoiled DNA and may not be suitable for downstream applications. Do not allow the lysis reaction to proceed longer than 5 minutes.

The HiPure kits should remove all protein from the DNA including endonucleases. For the silica-based PureLink® Quick Plasmid Miniprep Kit, we recommend an extra wash with the optional Wash Buffer W10 to remove endonucleases. This solution is not compatible with the HiPure system and should not be used with those kits. Alternatively, heat the eluted DNA in TE for 10 min at 70°C. This should heat-inactivate any contaminating nucleases.

Yes, we would recommend purchasing the PureLink® HiPure BAC Buffer Kit (Cat. No. K210018). You will need to add less RNase A than stated on the bottle label of the R3 buffer in this kit. It says to add 5.6 mL of RNase A. This is the correct amount for the BAC protocol; however, if you are performing standard plasmid isolation, 1.4 mL RNase A should be added.

A common problem encountered with absorbance measurements is turbidity of samples. (This could be caused by residual resin from the column.) If there is insoluble material in the cuvette (not often detected by the naked eye), much of the UV light is not absorbed but scattered, leading to an artificially high UV absorbance reading (at 260 or 280 nm, for example.) If your A260 is high, we recommend that you check the A320 to determine if there is resin in the sample. You can also try to centrifuge or filter (0.2 µm filter) your sample to remove any resin and then recheck the concentration.

We strongly recommend using the Elution Buffer provided in the kit, and do not recommend elution with water. If you need to elute in any other buffer, be sure to use a buffer of pH 8.5–9.0 for efficient DNA elution.

Yield can be variable, but should be between 500 µg and 1 µg per purification. This range is a result of: air pressure and air flow into the instrument, cell amounts (due to volume, strain, or type of medium), culture growth phase, plasmid size, and the ori on the plasmid.

The two most common seen causes for low/no yield are: low air pressure and/or flow rate (the instrument requires air flow rate of 4 cfm at 90 psi) and overloading (we recommend using 100–125 mL of LB culture at OD 2.0).

There are three possible reasons for this:
The variability of the air flow will reduce the amount of plasmid DNA recovered from the precipitator membrane. An expected elution volume recovery should be between 800 and 1,000 µl.

If larger plasmids are in use, the DNA size may impede the precipitator membrane enough to reduce the final elution flow. We have verified that elution volume (and yield) may be reduced if plasmids are large or exceed the recommended range.

If you using enriched medium (TB, Circlegrow®, etc.) then the low elution volume may be due to overloading the system, causing the pump to overload during the first few pumping cycles, causing blow-back into the reagent tray and blowing out some of the elution volume. A drop in house air pressure can also cause a low elution volume.

This is usually caused by a sudden dip in the air flow (an air system problem, usually not related to the instrument itself). During the BenchPro® 2100 procedure, the lysis step and elution step are the two steps that require higher air pressure, so this error message is usually seen at these two steps. If this error happens during the lysis step, we recommend restarting from the beginning, with a new culture, new card, and new reagent tray. There is no way to recover the sample.

If this happens at the elution step, you can see if you can get any eluted DNA. Hopefully some DNA has already eluted out (usually most DNA is eluted out in the first 1–2 minutes of the elution step). If this happens right before the elution step, with no solution in the elution tube yet, you can empty out the reagent tray reservoirs and refill them with 1.5 ml of TE or water in the right reservoir, then set up the run with the same tray and card and run the protocol again.

Please follow the sequence of steps below to try to release the drawer lock:

1. Power-off the instrument.
   
2. Remove card (if already inserted).
   
3. Press the drawer inward (holding the back of unit) to ensure the lock mechanism is not catching, then release pressure.
   
4. Power-on the instrument.
   
5. Press the drawer inward (holding the back of unit) again and attempt to open door.
   The software sends an unlock signal to the door lock solenoid during the start-up process. If after a few attempts of steps 1 through 5 the door still does not open, please call our Technical Support team.

It’s completely normal for this to happen during the run and we often see it, but haven't had it lead to any problems in the instrument. You can try widening the holes with the piercing tool, which will help any of the liquid run back into the tray. More debris on the top of the foil can occur when there is too much of a high OD culture added into the system; therefore, check this and scale back accordingly. After heavy use of the instrument, you may see some of this debris ending up in the drawer and occasionally on the tracks of the instrument, but it won’t hurt the instrument. However, if it's a concern, we would recommend taking some Kimwipes® wipers, moistening them with 70% ethanol, and wiping it down.