FAQs and Tech Support

Plasmid DNA Extraction- FAQs

Why does Viogene's Mini Plus^TM Plasmid DNA Extraction System require two washings with two types of wash buffers (WN and WS Buffer) while kits of some other brands (such as GFX Micro Plasmid Prep Kit from Amersham Pharmacia Biotech) just requires one washing?

WN Buffer is used to remove protein residues and degraded RNA residues on the membrane and WS Buffer is used to remove salt residues on the membrane. When a small volume of bacterial culture (less than 3 ml) is used, the lysate resulted is usually not rich in protein contaminants and so washing with only WS Buffer is already enough to result in plasmid pure enough for DNA sequencing and other applications. As one may notice that when a product kit only provides one wash buffer, it only allows purification of plasmid DNA from a culture of volume less than 3 ml. It is because one wash buffer is not enough to remove contaminants from a higher volume of culture. This kind of product only allows isolation of high copy plasmid as a small volume of culture is used. It cannot be used to isolate low copy plasmid as a higher volume of culture is required. Moreover, the drawback of using only one wash buffer is that it cannot remove degraded RNA bound to the membrane. Removal of RNA existing in the bacterial cells is achieved by degrading RNA released from cells by RNase added in MX1 Buffer. Degraded RNA does not bind well as undegraded RNA to the membrane in the presence of chaotropic salts, thus degraded RNA is washed off with wash buffer (WN Buffer) which contains chaotropic salts, whereas plasmid DNA is still bound to the membrane and is then eluted without RNA contamination. The single wash buffer provided in other kits does not contain chaotropic salts as WN buffer does, thus it is not able to remove degraded RNA bound to the column. In this case, degraded RNA will be co-eluted with plasmid DNA. Since RNA is degraded, an user does not see it by agarose gel electrophoresis analysis. Though degraded RNA does not affect restriction digestion and sequencing reaction, the presence of the ribo-oligonucleotides interferes with some applications such as digestion of plasmid with BAL 31 or labeling of the 5’ termini of restiction enzyme fragments of the plasmid with bacteriophage T4 polynucleotide kinase. Further, the presence of degraded RNA leads to a false high OD260 of the plasmid eluant (degraded RNA also absorbs light at wavelength of 260 nm), thus misleading the users that a high plasmid yield is obtained. The presence of degraded RNA in the plasmid DNA solution can be evidenced by OD260/OD280 ratio higher than 1.8. The use of two wash buffers provided in Viogene's kit solves these problems.

Can Mini Plus^TM plasmid DNA extraction system be applied on Gram(+) bacteria?

This system is mainly designed to extract plasmid DNA from Gram(-) bacteria such as E. coli. Gram(+) bacteria have thicker cell wall so cell lysis buffers provided in the kit does not lyse them readily. However, extraction of plasmid DNA from Gram(+) bacteria can still be achieved with additional treatment. After resuspending the pelleted bacterial cells in MX1 Buffer, add lysozyme to give a final concentration of 3 to 5 mg/ml. Incubate the suspension at 37˚C for 30-60 minutes (or for a shorter time when use 5 mg/ml lysozyme). This treatment weakens the cell wall of the Gram(+) bacteria. Then add MX2, and follow the rest of the protocol. For certain Gram(+) bacteria with thin cell wall, such as Lactobacillus, applying of double amount of MX1, MX2, and MX3 Buffer may have been enough to lyse the cells. Yet, we still recommend treating Gram(+) bacteria with lysozyme to facilitate cell lysis.

Why is there genomic DNA copurified with the plasmid?

When genomic DNA is found in the eluant, it means that genomic DNA has been sheared during cell lysis process. After MX2 Buffer is added, ensure that mixing is done very gently to prevent genomic DNA shearing. If genomic DNA is sheared, genomic DNA fragments will get into the lysate and copurified with plasmid.

Why do we get eluted plasmid contaminated with RNA?

Make sure that RNase A is added into MX1 Buffer. Store the MX1 Buffer at 4˚C. If RNase A-added MX1 Buffer is not properly stored at 4˚C or has been stored for a long time, e.g., more than 6 months, RNase A activity may have been reduced, thus not being able to degrade RNA completely. In this case, fresh RNase A has to added into MX1 Buffer with final concentration of 50 mg/ml. Again, store the buffer at 4˚C.

Why is it the eluted plasmid cannot be cut by restriction enzymes properly?

It is possible that salt residue in buffers or ethanol residue in WS Buffer is not removed completely and thus affects the downstream reaction. In case of salt residue, wash the column twice with WS Buffer. In case of ethanol residue, after washing with WS Buffer, make sure that the flow-through is discarded and centrifuge the column at full speed for 3 minutes. If necessary, to centrifuge for a few minutes more to ensure complete removal of ethanol.

Another reason is that plasmid is denatured. Denaturation happens if incubation in MX2 Buffer has gone for too long time. This can be visualized during electrophoresis that a band migrates faster than the supercoiled form. After MX2 Buffer is added, do NOT incubate for more than 5 minutes.

Plasmid DNA yield obtained by using Viogene's Mini Plus^TM Plasmid DNA Extraction System is not very consistent. For the same plasmid, why do we sometimes obtain a lower yield?

There are several possible reasons accounting for the lower plasmid yield obtained:

1) Bacterial culture did not grow well, thus resulting in a lower cell density. To ensure a well-grown culture, always inoculate bacterial cells from a freshly streaked plate and grow cells in the presence of the required antibiotic(s). Ensure that bacteria have grown well after overnight culture and attained an OD600 more than 1, meanwhile do not let the culture grow more than 16 hours, bacteria may enter death phase and plasmids in cells start to be degraded.

2) Do not use more than 5 ml of culture for one preparation. Sometimes when the culture is too dense, cells collected from a 5 ml culture cannot be completely lysed. Incomplete cell lysis will lead to a lower yield of plasmid.

3) If ddH2O of pH less than 7 is used for DNA elution, lower efficiency of plasmid elution will be resulted.

4) When a more concentrated plasmid DNA solution is desired, 30 ml of elution buffer is suggested. However, in comparison with using 50 ml elution buffer, there is about 40% of plasmid cannot be eluted when 30 ml is used. Therefore, no less than 30 ml of elution solution should be used.

5) Make sure that elution buffer is absorbed into the membrane and to stand the column for 1-2 minutes before centrifugation to elute DNA. If the buffer still retains on the membrane surface, plasmid DNA will not be eluted due to lack of contact with buffer. In this case pulse centrifugation of the column for 1-2 seconds (do NOT over-centrifuge) can help permeation of the buffer into the membrane.

6) Large plasmid is eluted less readily than small plasmid. When a plasmid is larger than 10 kb, use elution solution preheated to 70˚C.

Why is TB (terrific broth) not an ideal medium for culturing to prepare plasmids?

It is not recommended to use rich medium such as TB or 2X TY for most commonly used plasmids, especially high-copy number plasmids. Although rich medium have the obvious advantage of producing more bacteria, high level of cell biomass reduces the yield and quality of plasmids. If rich medium must be used, please reduce the culture volume to match the suitable range of cell biomass.

If the culture used too much, alkaline lysis will be inefficient to lower the yield of extracted plasmids. Furthermore, the excessive viscosity of the lysate will require vigorous mixing to result in shearing of genomic DNA and subsequent contamination of extracted plasmids. High level of protein and polysaccharide will usually be carried out with plasmids to result in bad quality of extracted plasmids.

To incubate bacterial cells with rich medium overnight (about 16 hours) is not recommended. Most of antibiotics will be ran out in 8 hours. The culture will lose the selection of the antibiotic and reduce the yield of plasmids in the bacterial cells.

Incubation with rich medium for long time, especially longer than 10 hours, will produce large amount of the dead cells. Sampling too many dead cells will provide low yield and bad quality of plasmids extracted.

Eluted plasmid appeared degraded after storage for a period of time.

When degradation appears, this indicates that nuclease the possible presence of nuclease in the eluted plasmid. There are several things to do:

1) Nuclease cannot be completely washed off especially when end+ E. coli host strain is used. Use end-strain if possible.

2) Wash the column twice with WF Buffer.

3) Use TE buffer for plasmid elution as EDTA can inhibit nuclease activity. Store eluted DNA at -20˚C when not used.

Tech Support

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