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1) Bacterial cells were not lysed completely: Too many bacterial cells were used. If more than 10 A600 units of bacterial culture were used, separate them into multiple tubes. After PD3 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 the PD Column matrix.
3) Incomplete DNA Elution: If plasmid DNA is larger than 10 Kb, use preheated Elution Buffer (60-70°C) in the 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 of elution buffer, there is about 40% of plasmid which cannot be eluted when 30 ml are used. Therefore, no less than 30 ml of elution solution should be used.
5) If ddH₂O, pH <7 is used for DNA elution, lower efficiency of plasmid elution will result.

1) Residual ethanol contamination: After the wash step, dry the PD Column with additional centrifugation at full speed for 5 minutes or incubate at 60°C for 5 minutes.

2) RNA contamination: Prior to using PD1 Buffer, ensure that RNase A was added. If RNase A added PD1 Buffer is out of date, add additional RNase A.

3) Too many bacterial cells were used: reduce sample volume.

4) Genomic DNA contamination: Do not use overgrown bacterial cultures. During PD2 and PD3 Buffer addition, mix gently to prevent genomic DNA shearing.

5) RNA contamination: Prior to using PM1 Buffer, check that RNase A was added. If RNase A added PM1 Buffer is out of date, add additional RNase A.

6) Too many bacterial cells were used: Reduce sample volume.

7) Genomic DNA contamination: Do not use overgrown bacterial culture samples. During the PM2 and PM3 Buffer addition steps mix solution gently to prevent genomic DNA shearing.

This system is mainly designed to extract plasmid DNA from Gram (-) bacteria such as E. coli. Gram (+) bacteria have thicker cell walls so the cell Lysis buffers provided in the kit do 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 the PD1 Buffer, add lysozyme to give a final concentration of 3 to 5 mg/ml. Incubate the suspension at 37ºC for 30-60 minutes (or for a shorter time when 5 mg/ml lysozyme is used). This treatment weakens the cell wall of the Gram (+) bacteria. Add PD2, and follow the rest of the protocol. For certain Gram (+) bacteria with thin cell walls, such as Lactobacillus, applying a double amount of PD1, PD2, and PD3 Buffer may be enough to lyse the cells. Yet, we still recommend treating Gram (+) bacteria with lysozyme to facilitate cell Lysis.

Make sure that RNase A is added into PD1 (or PM1) Buffer. Store the PD1 (or PM1) Buffer at 4ºC. If RNase A-added PD1 (PM1) Buffer is not properly stored at 4ºC 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 be added into PD1 (PM1) Buffer with a final concentration of 50 mg/ml. Again, store the buffer at 4ºC.

It is possible that salt residue in buffers or ethanol residue in the Wash Buffer is not removed completely, thus affecting the downstream reaction. In case of salt residue, 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, centrifuge for a few minutes more to ensure complete removal of ethanol. Another reason is that the plasmid is denatured. Denaturation occurs if incubation in PD2 Buffer is too long. This can be seen during electrophoresis. After PD2 Buffer is added, DO NOT incubate for more than 5 minutes.

W1 Buffer is used to remove protein residues and degraded RNA residues on the membrane and the 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 is usually not rich in protein contaminants so washing with only the Wash Buffer is already enough to result in plasmid pure enough for DNA sequencing and other applications. As one may notice, when a kit only includes one wash buffer, it only allows purification of plasmid DNA from a culture with a volume of less than 3 ml. This is because one wash buffer is not enough to remove contaminants from a higher volume of culture. This type of product only allows for 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 PD1 Buffer. Degraded RNA does not bind well to the membrane in the presence of chaotropic salts, thus degraded RNA is washed off with the 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 our 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, the user is unable to see it by agarose gel electrophoresis analysis. Though degraded RNA does not affect restriction digestion or sequencing reactions, 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 restriction 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 eluate (degraded RNA also absorbs light at wavelength of 260 nm), thus misleading the users, who assume 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 Geneaid's kit solves these issues.

When degradation appears, this indicates the possible presence of nuclease in the eluted plasmid. Refer to the following: (1) Nuclease cannot be completely washed off especially when end+ E. coli hoststrain is used. Use end-strain if possible. (2) Wash the column twice with W1 Buffer. (3) Use TE buffer for plasmid elution as EDTA can inhibit nuclease activity. Store eluted DNA at -20ºC when not used.

There are a few possible reasons that could lead to failure in restriction enzyme performance of extracted plasmid. (1) 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. (2) 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. (3) Plasmid Concentration: High concentrations of final extracted plasmid will cause similar problems.