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 If RNase A added PD1 Buffer is out of date, add additional RNase A.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 If RNase A added PM1 Buffer is out of date, add additional RNase A.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.
The Gel/PCR DNA Fragments Extraction Kit can only effectively remove (>90%) primers of less than 40 bp. When primers or dimer products are of more than 40 bp, they cannot be effectively removed. In this case, separate the PCR product from the dimer products by electrophoresis, excise the gel slice containing the desired product and purify it using Gel/PCR DNA Fragments Extraction Kit.
Yes, DF Buffer does not affect the chemically linked DIG on dNTP. Similarly, this system can be used to clean up 32P-labeled DNA fragments.
The smaller band may be a single-stranded form of the PCR product. The occurrence of it could be due to the elongation of the PCR product not being complete or the PCR product is denatured during the preparation. In this case, to re-anneal the single-stranded DNA, incubate the solution at 95ºC for 2 minutes and let it cool slowly to room temperature. The re-annealed PCR product can be used as usual in all downstream applications.
1) Gel slice did not dissolve completely: Gel slice was too big. If using more than 300 mg of gel slice, separate it into multiple tubes. Raise temperature of incubation to 60°C and extend incubation time.
2) Incorrect DNA Elution Step: Ensure that Elution Buffer was added and absorbed to the center of the DF Column matrix
3) Incomplete DNA Elution: If the sizes of DNA fragments are larger than 10 Kb, use preheated Elution Buffer (60-70°C) during the Elution Step to improve the elution efficiency.
4) Do not overload the column with too much DNA. Higher recovery is attained when lower amount of DNA is loaded. Divide the large amount of DNA into more than one column.
5) If ddH2O is used for elution, make sure that its pH is between 7.0 and 8.5, as pH lower than 7 leads to lower elution efficiency.
6) Make sure that complete DNA elution takes place by adding no less than 30 ml of elution solution onto the membrane and letting it completely absorb into the membrane before centrifugation.
7) Large DNA fragments are eluted less readily than small DNA fragments. When the DNA product is larger than 5 Kb, use the elution solution preheated to 60ºC.
1) Residual ethanol contamination: After the wash step, dry the DF Columnn with additional centrifugation at top speed for 5 minutes or incubate at 60°C for 5 minutes.
2) DNA was denatured (a smaller band appeared on gel analysis): Incubate eluted DNA at 95°C for 2 minutes, and then cool slowly to re-anneal denatured DNA.
1) Too much tissue was used: If using more than 20 mg of tissue, separate into multiple tubes.
2) Sample tissue was not lysed completely: Add additional Proteinase K and extend the incubation time in the Lysis step. After the Lysis step, centrifuge for 2 minutes at full speed (14,000 rpm) to remove sample debris. Transfer the supernatant to a new microcentrifuge tube and proceed with the DNA Binding Step.
3) Precipitate was formed at DNA Binding Step: Reduce the sample material. Before loading the column, break up the precipitate in ethanol-added lysate.
1) Sample tissue was not lysed completely: Add additional Proteinase K and extend the incubation time in the Lysis step.
2) Column was clogged at the DNA Binding Step: Following the Lysis Step, remove the insoluble debris by centrifugation. Prior to loading the column, break up the precipitate in ethanol-added lysate.
3) Incorrect DNA Elution Step. Ensure that Elution Buffer was added and absorbed to the center of the GD Column matrix.
4) Incomplete DNA Elution. Elute twice to increase the DNA recovery.
Several points should be noted to avoid DNA degradation: (1) DNA degradation occurs when the sample is not fresh or is stored improperly for a long time. Samples not used immediately should be flash frozen in liquid nitrogen and stored at -80°C. Genomic DNA in samples stored at room temperature, 4°C, or -20°C are subject to degradation. It is also not advised to keep samples in buffer or medium while storing at -80°C. (2) For whole blood samples, if they are stored at room temperature for more than 2 days or at 4°C or -20°C, isolated genomic DNA appears smeared at an extent proportional to the storage time. (3) Use fresh TAE or TBE running buffer for electrophoresis. Running buffer that is used repeatedly may be contaminated with DNase. (4) If isolated DNA needs to be stored for a long time, use 10 mM Tris-HCl (pH 9.0) or TE for elution. Using ddH2O is not advised in this case as DNA fragments in H2O suffer from gradual degradation through acid hydrolysis. (5) If DNA is to be used frequently, elute in 10 mM Tris-HCl (pH 9.0) or TE and store at 4°C. Keep DNA at -20°C only for long-term storage. Repeated freeze-thaw cycles can cause shearing of genomic DNA. (6) Genomic DNA extracted from paraffin-embedded tissue is usually degraded. This is because genomic DNA in paraffin-embedded tissue unavoidably suffers from degradation when samples are treated and stored for a long time. DNA in this case is not suitable for Southern blotting or restriction analysis due to smearing. However, it is applicable for PCR.