A-1 Ethanol was not added to the washing buffer

----Make sure that ethanol has been added to Buffer PW before use.

A-2 Ethanol remains on the adsorption material

----There is washing buffer residue on the silica membrane during elution, which will reduce the elution efficiency due to the presence of ethanol. The washing buffer can be completely removed by centrifugation or by placing the column in a 50°C incubator for 5-10 min before elution.

A-3 The elution buffer pH is low

----DNA can only be eluted in low salt buffers, such as Buffer EB (10 mM Tris-Cl, pH 8.5) or water. The elution efficiency depends on the pH value, and the maximal elution efficiency can be obtained when the pH is between 7.0 and 8.5. Please ensure the pH value within this range when eluted with water.

A-4 Elution buffer is not added to the correct location

----The elution buffer should be added to the center of the silica gel membrane to ensure that the buffer can completely cover the surface of the silica membrane for maximal elution efficiency.

A-5 Initial DNA amount is too low

----In order to get the recovery of more than 1 μg DNA, first enrich DNA fragments to ensure efficiency.

A-6 The volume of Buffer PB is not added correctly

----Please strictly add 5 times of buffer PB to the initial sample amount.

A-1 Gel is not completely dissolved.

----Dissolve the gel in a 55 ℃ water bath and constantly convert the tube up and down to mix, to ensure the gel is completely dissolved before proceeding to the next step. If the gel concentration is high, please increase volume of the gel lysis buffer.

A-2 Gel block is too large (>400 mg).

----If the gel block to be treated is too large, please cut it into small pieces, and divide the recovery into twice or choose a maxi kit.

A-3 The pH value of electrophoresis buffer is too high.

---DNA will bind to the silica membrane at high salt and low pH (pH≤7.5) condition and will be eluted out at low salt and high pH (pH≥8) condition. If the pH of the running buffer is too high, it will result in no DNA binding or low binding rate. It is recommended to add 10 μl NaAc (pH5.0) after the gel lysis to adjust the pH to below 7.5.

A-4 Ethanol not added in the washing buffer.

----Ethanol should be added to the washing buffer before the first use. The bottle containing washing buffer should be sealed after each use to avoid evaporation of ethanol and the reduction of recovery rate.

A-5 Incorrect elution buffer

----DNA can only be eluted in low salt solutions, such as Buffer EB (10 mM Tris-Cl, pH8.5) or water. The elution efficiency depends on the pH value, and the maximal elution efficiency can be obtained when the pH is between 7.0 and 8.5. When eluting with water, make sure that the pH is within this range.

A-6 Elution buffer is not added in the middle of the adsorption membrane.

----The elution buffer should be added in the middle of the adsorption membrane in the spin column, especially for small amount of buffer. Incubate for 1-2 min, and collect the eluate by centrifuging.

A-7 Low amount of DNA fragments.

----Make sure the DNA starting amount is greater than 1 μg, if not, please enrich the fragments first.

A-8 The volume of Buffer PN is not correct

----Please add the equal volume of Buffer PN as the actual volume of gel blocks.

A-1 Ethanol in the eluate

----There is washing buffer residue on the silica membrane or silica resin during elution, which will result in the remaining of ethanol in the eluate, and downstream operations will be affected. Re-centrifuge the column or incubate in a 50℃ incubator for 5-10 min to thoroughly remove the washing buffer before elution step.

A-2 Agarose in the eluate

----The gel is not completely dissolved.

A-3 The eluate contains ssDNA, which appears as a small smear of bands on the agarose gel.

----Heat the eluate to 95℃ for 2 min, and slowly cool to room temperature to re-anneal the single-stranded DNA.

Ethanol remains in the eluent

----There is washing buffer residue on the silica membrane during elution, which will reduce the elution efficiency due to the presence of ethanol. The washing buffer can be completely removed by centrifugation or by placing the column in a 50°C incubator for 5-10 min before elution.

A-1 E.coli aging

——In general, the glycerol preservation bacteria strains need to be activated before inoculation.

——Select new colonies from the plate culture into the liquid seed culture, and inoculate 1/1000 seed culture to the overnight culture medium.

A-2 Low plasmid copy number

——The low yield of plasmid DNA might be caused by the low copy number vectors. The yield can be increased by changing to a high copy number vector.

A-3 No plasmid in the bacteria

——Some plasmids are not stable in certain bacteria strains and may result in plasmid loss after multiple inoculations. For example, cosmids are unstable in E.coli for long-term storage. So do not inoculate frequently using the same bacteria seed, and each time please inoculate a single colony. In addition, check if the concentration of antibiotics used in screening is correct.

——Excessive shaking time might cause the death of the bacteria. For example, E.coli containing pUC18 should not be shaking cultured for more than 14 h.

A-4 Alkaline lysis was inefficient

——Applying too much bacterial culture medium in the extraction will result in insufficient cell lysis, which can be avoided by reducing the amount of bacteria or increase the volume of Buffer P1, P2 and P3. For low-copy plasmids, if the bacteria culture medium exceeds 5 ml, the volume of Buffer P1, P2 and P3 should be doubled to increase the yield and quality of plasmids.

A-5 Buffer incorrectly prepared

——Cloudy precipitates might appear in Buffer P2, P3 at low temperature. Please place them at 37℃ until the buffer turns clear before applying on the plasmid extraction.

A-6 Overloading of the adsorption column

——The adsorption capacity of the adsorption column is different for different products. For the extraction of large amount plasmid, please separate the extraction into several columns. If enrichment medium, such as TB or 2x YT are used, the volume of the bacteria medium should be reduced. If the plasmid or host strain is of very high copy number or growth rate, the volume of the LB medium must be adjusted.

A-7 Plasmid is not completely eluted

——Heat the column appropriately or prolong the incubation time for the elution step.

A-8 Ethanol residue in the plasmid

——Extend the centrifugation time after the washing step to remove the buffer as much as possible. Add the elution buffer into the dried column.

A-9 The elution buffer hasn’t completely cover the membrane

——The elution buffer should be added to the center of the silica gel membrane to ensure that the buffer will completely cover the surface of the silica membrane, which can maximize the elution efficiency.

A-10 Incorrect elution buffer

——DNA can only be eluted in low salt solutions, such as Buffer EB/TB (10 mM Tris-Cl, pH 8.5) or water. The elution efficiency depends on the pH. The maximum elution efficiency is between pH 7.0 and 8.5. When eluting with water, ensure that the pH is within this range. The yield will decrease if the pH is too low. The elution efficiency can be increased by heating the elution buffer or sterilizing distilled water to 60℃ .

A-11 Elution volume is too low

——The elution volume has an effect on the recovery rate. As the elution volume increases, the recovery rate increases, but the plasmid concentration decreases. The recovery rate can be increased by increasing the elution volume.

A-12 The lysis time is too long

——The lysis time should not exceed 5 min after adding Buffer P2.

A-13 The elution time is too short

——The elution time also have an effect on the recovery rate. Incubate the elution buffer with the plasmid before centrifuging can lead to better results.

A-1 Proteins in the eluate

——Do not use too much bacteria culture medium. After the treatments of Buffer P1, P2 and P3, the solution should  be clear. If microprotein suspension is present in the solution, centrifuge the solution to remove the suspension before proceeding to the next step.

A-2 RNA in the eluate

——Reduce the bacteria sample amount or leave at room temperature for a while after adding Buffer P3. If RNase A has been added into Buffer P1 for more than 6 months, re-add RNase A in Buffer P1 to make a new mix.

——For the Maxi kits, strictly control the amount of isopropanol.

A-3 Genomic DNA in the eluate

——The solution should be mixed gently after the addition of Buffer P2 and P3. If mix vigorously, the genomic DNA may be sheared into fragments and mixed with the plasmids. If the solution become too viscous and can not be mixed gently after adding Buffer P2, please reduce the amount of bacteria. Prolonged bacteria culture time will lead to degradation of cells and DNA, so the incubation time should not be more than 16 h.

A-4 The solution has been placed too long after the addition of Buffer P3

——The solution should not be placed too long after the addition of Buffer P3, otherwise there might be small DNA fragments contamination.

A-5 Host bacteria containing a large number of nucleases

——Some host bacteria contain a large number of nucleases, which degrade the plasmid DNA during plasmid extraction and affect the integrity of the extracted plasmid DNA. The best solution is to use a nuclease-free E.coli host stain such as DH5α or Top 10.

——Apply the deproteinized solution Buffer PD to remove residual nuclease.

A-6 Ethanol in the eluate

——Extend the centrifugation time after the washing step to remove the buffer as much as possible. Add the elution  buffer into the dried column.

A-7 Dimer and multimer forms of plasmids

——Dimers and multimers can be formed during plasmid replication, which depends on host bacteria strains. They can be detected by electrophoresis, but will not affect downstream experiments such as restriction endonuclease digestion, transformation, sequencing, etc.

G⁺ bacteria should be treated with lysozyme to break the cell wall. The treatment method is as follows: harvest appropriate amount of bacteria, and add 250 μl of Buffer P1 to resuspend the bacterial pellet, after that, add lysozyme to the final concentration of 10-20 mg/ml. Incubate at 37℃ for 30 min. The concentration of lysozyme added and the treatment time can be adjusted according to the bacteria strain and the specific experimental conditions. Please refer to the handbook for the following procedure.