FAQ: RPM Kits

My cells didn't lyse completely. Why not?

Does the way I grow my overnight affect my yield with the RPM Kit?

Given that I have plasmid in my culture, what could have gone wrong in the RPM protocol that might lead to a low yield?

I was doing a large scale plasmid prep and recovered DNA after elution, but seemed to lose it in the precipitation. What could have happened?

I seem to have chromosomal DNA contamination. Why?

When I ran my prep on a gel, I had more than one band. Why?

I have RNA contamination in my prep. How did it get there and how do I get rid of it?

(RPM 1G, 4G, and 12G only) I'm having trouble resuspending my precipitated DNA on the last step. Why?

Q. My cells didn't lyse completely. Why not?

A. There are at least two possible reasons:

1. The detergent in the Alkaline Lysis Solution precipitated. If the Alkaline Lysis Solution (ALS II in the RPM 4G/12G kits) is stored at ‹15°C, the detergent in the solution could precipitate, lowering the concentration of the detergent and causing poor lysis. If a precipitate is present, heat the solution to 60°C while mixing until detergent is resuspended.

2. Too many cells were processed. The number of cells in a culture can vary due to several factors, such as volume, growth medium, aeration, host cell strain, concentration of antibiotic, etc.. When the number of cells is too great, the volume of the lysis solution may be insufficient for complete lysis. If the pellet weight is greater than the value recommended in the protocol, either dilute the culture or increase the volumes of the Pre-Lysis, Alkaline Lysis and Neutralizing Solutions proportionally. In the binding step, add 5.5 M guanidine thiocyanate to the Glassmilk Spinbuffer (or RPM XG Glassmilk) so that the amount of GTC Glassmilk Spinbuffer/RPM XG Glassmilk is equal to the volume of Pre-Lysis Alkaline Lysis Neutralizing Solution.

Q. Does the way I grow my overnight affect my yield with the RPM Kit?

A. When growing the cells, it is important to maintain selection for the plasmid. If there are low concentrations of antibiotic or if the antibiotic has lost potency, even cells lacking plasmid will be able to grow. These cells are able to replicate at a faster rate than cells containing the plasmid, so even though you may have a large cell pellet, you will get a low plasmid yield. Also, optimal growth conditions need to be maintained for good growth (i.e. incubation temperature, aeration, etc.). Failure to maintain optimal conditions could result in poor growth.

Q. Given that I have plasmid in my culture, what could have gone wrong in the RPM protocol that might lead to a low yield?

A. The RPM protocol consists of a modified alkaline lysis followed by a GENECLEAN procedure. Both procedures have been used for years in the DNA isolation process and have been refined to the point where there are only a few steps where a problem is likely to occur.

• If the overnight culture was ok, the next problem could be the lysis step (see first question in Q & A). Poor lysis could lead to inefficient release of the plasmid DNA.
• In the wash step, if the Wash Solution's ethanol concentration drops significantly, the DNA may elute in the wash. DNA may also elute in the wash if ethanol was not added to the solution prior to use.
• In the elution step, if the Glassmilk/DNA complex was not resuspended, the DNA will not elute efficiently, leading to low recoveries.

Q. I was doing a large scale plasmid prep and recovered DNA after elution, but seemed to lose it in the precipitation. What could have happened?

A. For the larger RPM kits (RPM 1G, 4G, 12G), the DNA could be lost in the isopropanol precipitation step. After adding the NaCl and isopropanol, incubate the solution at -70°C for 10-30 minutes. Mark the side of the centrifuge tube where you expect the pellet to be since it may be difficult to visualize. When resuspending the pellet in water, be sure to check the sides of the centrifuge tube for residual DNA (if you're using a fixed-angle centrifuge) and that the pellet is completely dissolved before quantifying your results.

Q. I seem to have chromosomal DNA contamination. Why?

A. Rough handling of the cells during lysis (i.e. vortexing, vigorous mixing, pipetting) or leaving the DNA in the Lysis Solution for ›5 minutes can lead to chromosomal DNA contamination. If working with the RPM 4G or 12G kits, use the Modified High Copy Protocol.

Q. When I ran my prep on a gel, I had more than one band. Why?

A. When doing a plasmid prep, the plasmid DNA can appear in many conformations.

• Supercoiled: This is the optimal band for plasmid preps. This DNA band appears to run faster in the gel than the linear band and under optimal conditions, should be the lowest band on the get.
• Denatured Supercoiled: Extended incubations in the Alkaline Lysis Solution can cause the formation of denatured supercoiled DNA, which will run faster than supercoiled plasmids. This band can usually be eliminated by shortening the lysis of incubation (‹5 minutes) or, if working with the 4G or 12G kits, by reducing the amount of Alkaline Lysis Concentrate II by 10-20%
• Nicked or linearized DNA: These forms of DNA will run slower than the supercoiled forms. Nicked or linearized DNA can also be formed by rough treatment of the DNA/silica pellet during the binding and wash steps. Gently resuspending the pellets should reduce the amount of these forms of plasmid DNA.
• Dimers and multimers: These run slower and are sometimes confused with nicked, linearized, or chromosomal DNA. Theire presence is dependent upon host strain and growth medium used.For RPM 4G and 12G only:

Q. I have RNA contamination in my prep. How did it get there and how do I get rid of it?

A. 1. The RNA may be present due to insufficient RNase digestion. If using the High Copy Protocol, add an additional 20-50 µl of RNase Mixx at the Pre-Lysis step and/or increase the incubation period. Alternatively, add the additional RNase Mixx after clearing the lysate following the neutralization step, then incubate for 15-30 minutes in a 55°C water bath.

2. When working with the Modified High Copy/Low Copy protocol, the RNA may not have precipitated after the LiCl treatment. After adding the LiCl, incubate on ice for 15 minutes before centrifugation.

3. The RNase digestion may not have been complete. Add 20-50 µl of RNase Mixx to the solution after clearing the lysate from the neutralization step. Increase the 55°C incubation time.

Q: (RPM 1G, 4G, and 12G only) I'm having trouble resuspending my precipitated DNA on the last step. Why?

A. Two possibilities:

1. The pellet is overdried. After adding the water, incubate the tube in a 37°C water bath until the DNA dissolves.

2. The pH of the solution used to resuspend the DNA pellet after precipitation is too low. DNA dissolves better in slightly alkaline conditions.