Best Plasmid Purification: 10 Reasons Your Yields Are Low

Good yields are vital in plasmid isolation. How do you optimize your preparation to achieve a good plasmid yield and what causes low yields? We address these questions and much more below:

What is the best yield during plasmid isolation?

There are three main types of plasmid preparation: mini, midi and maxi. The amount of plasmid DNA varies between protocols and kits, but the table below provides an average range for these three different preparation methods.

**When both samples are processed at the same time and in the same way, if one sample yields well and the other plasmid yields poorly, what could be the problem? Many things can cause yield differences between plasmid preparations. Let’s unravel this mystery item by item and see what we can do.

You prepared two plasmids at the same time using the same protocol and got different yields. If the plasmids have the same “backbone” then the cause could be an insert. Some inserts can be problematic for bacteria, for example a protein that makes them sick, or unstable, such as repetitive sequences. You can try using specialized cell lines to overcome the problem.

2. Number of Copies

The second important point is how the insert size changes the copy number of the plasmid. Large inserts will reduce the copy number of the plasmid, meaning you will need to grow more cells to get a good yield.

If genes are cloned into different vectors, the problem could be that the plasmids are copied at different rates. One might be a high-copy plasmid, another might be a medium or even low-copy plasmid.

3. Culture Saturation

Good results are always obtained when preparing the culture. Inoculation from old colonies or over-saturation of the culture will severely negatively affect the plasmid yield. We do not want cultures to be in the late latency phase and we do not want them to be over-saturated.

4. Using Old Colonies

The age of the petri used for starting cultures is important. If your petri is old, even if you have selected a nice big colony, not all of them will be viable cells. Therefore, for best results, purify on a new petri before you start.

5. Antibiotic Problems

Another cause of weak plasmid is antibiotics. Bacteria break down antibiotics as they grow in culture. If not enough antibiotics are added, or if the stocks are old and underpowered, the antibiotic may not be so dominant and you may end up with an antibiotic-free culture.

6. Lysis and Neutralization Problems

Some reagents in lysis are mostly good and stable. But when exposed to air, they can deteriorate over time and may not work as well as on the first day.

One of the biggest mistakes workers make in plasmid isolation is lysis. While protocols normally tell you to be gentle, it’s not good to be too precise.

Another common problem is that lysis is allowed to continue for too long, resulting in permanently denatured, indigestible DNA.

7. Isopropanol Quality

Many laboratories have large bottles of isopropanol that are opened and closed throughout the year. If we want to get good yields from the precipitation step, you have to make sure that the isopropanol used is not old.

8. Losing the Pellet

Isopropanol pellets are glassy and clear and difficult to see. A best practice would be to mark the spot where you expect the pellet to form after centrifugation in a fixed angle rotor. This way you know where to find the plasmid when you pour the isopropanol.

Another way to ensure that you know where your pellet is when using a fixed angle rotor is to always load your tubes in the same way, so your pellet will always be in the same place.

!!! Note that many commercial kit manufacturers have developed kits for this problem, using “precipitators” or silica disk filters.

!!! Also a small note: we recommend not to reduce the centrifugation time and speed shorter than optimized. If you cannot increase the centrifugation speed, never shorten the time.

9. Blockage of Columns

If you are worried about clogging the columns, strain through Whatman paper before proceeding to the column step.

10. DNA Remaining in Columns

DNA may remain on the columns after elution. Perform a separate elution to remove any remaining DNA. Using elution buffer heated to around 50°C can also help increase elution efficiency.

References:

Maniatis, T., Fritsch, E. F. and Sambrook, J. Molecular cloning. New York: Cold Spring Harbor Laboratory; 1982. pp 545.

Begbie S. et al. (2005) The Effects of Sub-Inhibitory Levels of Chloramphenicol on pBR322 Plasmid Copy Number in Escherichia coli DH5? . Journal of Experimental Microbiology andImmunology (JEMI). 7:82-8.