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 PD1 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 PD2, and follow the rest of the protocol. For certain Gram(+) bacteria with thin cell wall, such as Lactobacillus, applying of double amount of PD1, PD2, and PD3 Buffer may have been enough to lyse the cells.Yet, we still recommend treating Gram(+) bacteria with lysozyme to facilitate cell lysis.

W1 Buffer is used to remove protein residues and degraded RNA residues on the membrane and Wash 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 therefore washing with only Wash Buffer is already sufficient to achieve plasmid purity enough for DNA sequencing and other applications . Users 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 sufficient to remove contaminants from a higher volume of culture. This kind of products only allows isolating high-copy plasmid as a small volume of culture is used. It cannot be applied 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 PD1 Buffer. Degraded RNA does not bind as well as undegraded RNA to the membrane in the presence of chaotropic salts, th us degraded RNA is washed off with wash 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 Wash 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, users do 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 OD 260 of the plasmid eluent (degraded RNA also absorbs light at wavelength of 260 nm), thus misleading users that a high plasmid yield is obtained. The presence of degraded RNA in the plasmid DNA solution can be evidenced by OD 260 /OD 280 ratio higher than 1.8. The use of two wash buffers provided in Geneaid's kit solves these problems.

Unsatisfied performance can be caused by a number of different factors:

( 1) Residual ethanol contamination

After wash step, dry PD Column with additional centrifugation at top speed for 5 minutes or incubation at 60 ℃ for 5 minutes.

(2) RNA contamination

•  Prior to using PD1 (or PM1) Buffer, ensure that RNase A was added. If RNase A added PD1 (or PM1) Buffer is out of date, add additional RNase A.

•  Too many bacterial cells were used, reduce sample volume.

(3) Genomic DNA contamination

•  Do not use overgrown bacterial culture.

•  During PD2 (or PM2) and PD3 (or PM3) Buffer addition, mix gently to prevent genomic DNA shearing

(1) It is possible that salt residues in buffers or ethanol residues in Wash Buffer are not removed completely and thus affect the downstream reaction. In case of salt residues, wash the column twice with Wash Buffer. In case of ethanol residue, after washing with Wash 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.

(2) Another possibility is that plasmid is denatured. Denaturation happens if incubation in PD2 Buffer has been for too long time. This can be visualized during electrophoresis that a band migrates faster than the supercoiled form. After PD2 Buffer is added, do NOT incubate for more than 5 minutes.

(3) There are still other possibilities that could lead to fail in restriction enzyme performance of extracted plasmid:

•  Enzyme Activity: most restriction enzymes are temperature sensitive, prolonged storage or usage past expiry date will decrease enzyme activity dramatically, hence causing partial or total failure of plasmid cutting.

•  Sensitivity: different strains of E. Coli will have varying sensitivity to dam or dcm methylation, if digestion sites were blocked by overlapping dam or dcm methylation during transformation, it will cause difficulty at the restriction enzyme digestion step.

•  Plasmid Concentration: high concentrations of final extracted plasmid will cause similar problems.

•  Supercoiled form: high quality plasmid extraction kits always get high yields of supercoiled form of plasmids, please refer to the instruction of restriction endonucleases that customer use. 

( 1) Bacterial cells were not lysed completely

•  Too many bacterial cells were used. If use more than 10 A 600 units of bacterial culture, separate it into multiple tubes.

•  After PD 3 Buffer addition, break up the precipitate by inverting to ensure higher yield.

(2) Incorrect DNA Elution Step

•  Ensure that Elution Buffer was added and absorbed to the center of PD Column matrix.

(3) Incomplete DNA Elution

•  If plasmid DNA is larger than 10 kb, use preheated Elution Buffer (60 -70 ℃ ) on Elution Step to improve the elution efficiency.

(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) If ddH 2 O of pH less than 7 is used for DNA elution, lower efficiency of plasmid elution will be resulted.

When degradation appears, this indicates that 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.