(1) Possibly some precipitate formed during the DNA precipitation step, which can be resolved by reducing the sample volume and increasing standing time to improve DNA precipitation. (2) Increase incubation time following Isopropanol addition to improve total Nucleic Acid precipitation. (3) Avoid RNase contamination.
Most likely too much sample was used. (1) Reduce the sample volume or separate the sample into multiple tubes.
The size of plasmid affects the efficiency greatly. For instance, the efficiency for a supercoiled 2.7 Kb and a 10 Kb plasmid (using 1 minute protocol and DH5α-CD191) is 1.6~5.5 x 109 and 4.0~9.0 x 106 respectively. The difference is approximately 100 times. Transformation efficiency for large plasmids (especially > 6 Kb) can be increased by using the optional protocol as described in Q4.
For large plasmid (> 6 Kb), the optional protocol will significantly improve the efficiency.
There is little difference between 15~45 seconds of heat-shock. For most Elite™ strains, 15~35 seconds of heat-shock is optimal for transformation of plasmid < 6 Kb. 45 second heat shock is not optimal for plasmid < 6 Kb (may decrease 1.2~2.5 times, strain dependent) but is optimal for > 6 Kb plasmid.
Slow thawing or unstable freezing will cause decreases in efficiency. Therefore, it is very important that the competent cells are stored at -70ºC at all times. Thawing the competent cells in room temperature water yields better efficiency opposed to thawing the cells on ice.
The Presto™ Mini RNA Yeast Kit.
The extra filter columns are effective in clearing cell debris and ensuring full cell Lysis.
For isolation of Cytoplasmic RNA from animal cells or eukaryotic cells, the Total RNA Mini Kit (Blood/Cultured Cell) will satisfy this requirement. Extra buffer (not included in the kit) will have to be prepared to lyse the plasma membrane before proceeding with the regular protocol. Plasma Lysis Buffer Components: (Pre-cool to 4°C) 50 mM Tris-Cl, pH 8.0 140 mM NaCl 1.5 mM MgCl2 0.5% (v/v) Nonidet P-40‡ (1.06 g/ml). Just before use, add: 1,000 U/ml RNase inhibitor 1 mM DTT. Add Buffer to lyse plasma membrane: For pelleted cells, loosen the cell pellet thoroughly by flicking the tube. Carefully resuspend cells in 175 µl cold (4°C) Plasma Lysis Buffer, and incubate on ice for 5 minutes.
No, some genomic DNA (and plasmid DNA, if present) can be co-purified with RNA. DNA can be removed by adding RNase-free DNase I to the RNA sample. DNase I can then be removed by phenol/chloroform extraction.
Three critical steps, if not performed well can cause RNA degradation. Handling and storing of samples, disruption of samples and storage of eluted RNA. (1) Most animal tissues can be processed fresh (unfrozen). It is important to keep fresh tissue cold and to process it quickly (within 30 minutes) after dissecting. If samples cannot be processed immediately, it should be flash frozen in liquid nitrogen and stored at -80°C. Samples should be handled with RNase-free tools. (2) When the sample is disrupted, disruption needs to be fast and thorough. Slow disruption (e.g. placing cells or tissue in RB Buffer without any additional physical shearing) may result in RNA degradation by endogenous RNase released internally, yet still inaccessible to the protein denaturant in the buffer. (3) After elution of RNA with RNase-free ddH2O, store RNA at -80°C. (4) Degradation of RNA may also occur during loading into a gel. Use gel and fresh running buffer prepared using DEPC-treated ddH2O, as well as a properly cleaned geltray and tank for electrophoresis. Adding EtBr directly into the gel can also avoid possible degradation of RNA that may occur during gel staining.
(1) Poor yield of total RNA is mostly due to incomplete sample Lysis, thus leading to incomplete release of RNA. Since good yield and good quality of total RNA are only assured when the sample is properly handled and lysed completely, DO NOT use more than the amount of sample suggested in the protocol. (2) Thorough cellular disruption is critical for high RNA quality and yield. RNA that is trapped in intact cells is often removed with cellular debris and is unavailable for subsequent isolation. Therefore, it is crucial to choose the disruption method best suited to a specific tissue or organism to maximize yield. Mechanical cell disruption techniques include grinding, homogenization, vortexing, sonication etc. Complete disruption of some tissues may require using a combination of these techniques. (3) Another, more common cause of low RNA yield is overloading the column, which can cause the column to clog or can prevent the RNA from binding to the membrane efficiently. Methods that reduce viscosity, such as reducing sample amount, disrupting the sample more extensively, and centrifuging to remove insoluble remains, will increase RNA yield. If yields are still lower than expected, consider diluting the clarified lysate and splitting loading into two columns, which will further reduce the concentration of contaminants and improve RNA binding and recovery. (4) When RNA is to be eluted, make sure that RNase-free ddH2O is added onto the membrane and absorbed completely. If ddH2O still remains on the membrane, pulse centrifuge the column for a few seconds to drag it into the membrane.
This indicates that the number of WBC (white blood cells) in the Buffy coat is too high, thus not being lysed and digested completely by Proteinase K. Buffy coat should be prepared from a lower volume of whole blood and make sure that fewer than 1x107 of WBC is used per preparation. Incubation should be done with constant mixing to disperse Proteinase K and sample. If Lysis is incomplete, add more Proteinase K and repeat incubation. The sample should not contain insoluble residues when it is completely digested. Centrifuge to remove any undigested residues and only use the supernate to continue the procedure.
If a sample is rich in protein, complete digestion will not be achieved using the amount of Proteinase K and buffer suggested in the protocol. If a sample cannot be digested completely or appears very viscous, add more Lysis Buffer and repeat incubation. Centrifuge the sample at full speed for 5 minutes to remove undigested remains and only use the supernatant in the following steps. In the subsequent preparations, a lower amount of the sample should be used. A general rule of thumb is to start with half of the maximum amount of sample suggested. When there isn’t a problem in digesting the sample completely or passing the lysate through the column, the sample amount can be increased gradually in the subsequent preparations.
The key is to use fresh samples and not to overload the column. Low yield or purity of genomic DNA is usually due to incomplete digestion or incomplete Lysis of the sample. Starting with a maximum amount or volume of samples does NOT usually give the best yield of DNA. On the contrary, it usually results in incomplete sample Lysis and degradation of proteins, thus making extraction of all DNA from the sample unfeasible. Further, it always requires subsequent removal of undigested residues and yields viscous sample lysate. When the lysate is too viscous, it not only has difficulty in passing the column, but also indicates the presence of an abundant amount of contaminants such as proteins and salts. High amounts of contaminants not only affect DNA binding, but also may not be washed off completely, leading to carry over to the eluted genomic DNA. Therefore, a good quality and yield of DNA is only expected when a sample is completely digested. We advise starting with half of the maximum amount of sample suggested. When there aren’t any problems with digestion or passing the lysate through the column, the sample amount can be increased gradually in the subsequent preparations.
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.
(1) Residual ethanol contamination: Following the wash step, dry the GD Column with additional centrifugation at full speed for 5 minutes or incubate at 60°C for 5 minutes. (2) RNA contamination: Perform Optional RNA degradation Step. (3) Protein contamination: Reduce the sample amount. After the DNA Binding Step, apply 400 ml W1 Buffer to wash the GD Column and centrifuge at 13,000 rpm for 30 seconds. Proceed with the Wash Step. (4) Genomic DNA was degraded. Use fresh samples or freeze fresh samples in liquid nitrogen immediately and store at -80°C.
(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.
(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) 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) 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.
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.
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 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.
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.
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.
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.
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.
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.
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.
(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. (4) Too many bacterial cells were used: reduce sample volume. (5) Genomic DNA contamination: Do not use overgrown bacterial cultures. During PD2 and PD3 Buffer addition, mix gently to prevent genomic DNA shearing. (6) 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. (7) Too many bacterial cells were used: Reduce sample volume. (8) 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.
(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.