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Why is TB (terrific broth) not an ideal medium for culturing to prepare plasmids? |
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It is not recommended to use rich medium such as TB or 2X TY for most commonly used plasmids, especially high-copy number plasmids. Although rich medium have the obvious advantage of producing more bacteria, high level of cell biomass reduces the yield and quality of plasmids. If rich medium must be used, please reduce the culture volume to match the suitable range of cell biomass.
If the culture used too much, alkaline lysis will be inefficient to lower the yield of extracted plasmids. Furthermore, the excessive viscosity of the lysate will require vigorous mixing to result in shearing of genomic DNA and subsequent contamination of extracted plasmids. High level of protein and polysaccharide will usually be carried out with plasmids to result in bad quality of extracted plasmids.
To incubate bacterial cells with rich medium overnight (about 16 hours) is not recommended. Most of antibiotics will be ran out in 8 hours. The culture will lose the selection of the antibiotic and reduce the yield of plasmids in the bacterial cells.
Incubation with rich medium for long time, especially longer than 10 hours, will produce large amount of the dead cells. Sampling too many dead cells will provide low yield and bad quality of plasmids extracted. |
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How do I increase the recovery of the DNA fargment with small or large size? |
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<For Gel Extraction>
Add 0.25 volume of isopropanol of the mixture from STEP 3. and mix well. That will increase the recovery of the DNA, especially when the size of the DNA fragment is < 500-bp or > 5-kbp.
<For PCR Clean up>
Add 0.25 volume of isopropanol of the mixture from STEP 1. and mix well. That will increase the recovery of the DNA, especially when the size of the DNA fragment is < 500-bp or > 5-kbp. |
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| 3 |
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Plasmid DNA yield obtained by using Viogene's Mini-M® Plasmid DNA Extraction System is not very consistent. For the same plasmid, why do we sometimes obtain a lower yield? |
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There are several possible reasons accounting for the lower plasmid yield obtained:
1) Bacterial culture did not grow well, thus resulting in a lower cell density. To ensure a well-grown culture, always inoculate bacterial cells from a freshly streaked plate and grow cells in the presence of the required antibiotic(s). Ensure that bacteria have grown well after overnight culture and attained an OD600 more than 1, meanwhile do not let the culture grow more than 16 hours, bacteria may enter death phase and plasmids in cells start to be degraded.
2) Do not use more than 5 ml of culture for one preparation. Sometimes when the culture is too dense, cells collected from a 5 ml culture cannot be completely lysed. Incomplete cell lysis will lead to a lower yield of plasmid.
3) If ddH2O of pH less than 7 is used for DNA elution, lower efficiency of plasmid elution will be resulted.
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) Make sure that elution buffer is absorbed into the membrane and to stand the column for 1-2 minutes before centrifugation to elute DNA. If the buffer still retains on the membrane surface, plasmid DNA will not be eluted due to lack of contact with buffer. In this case pulse centrifugation of the column for 1-2 seconds (do NOT over-centrifuge) can help permeation of the buffer into the membrane.
6) Large plasmid is eluted less readily than small plasmid. When a plasmid is larger than 10 kb, use elution solution preheated to 70˚C. |
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Why does Viogene's Mini-M® Plasmid DNA Extraction System require two washings with two types of wash buffers (WF and WS Buffer) while kits of some other brands (such as GFX Micro Plasmid Prep Kit from Amersham Pharmacia Biotech) just requires one washing? |
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WF Buffer is used to remove protein residues and degraded RNA residues on the membrane and WS 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 so washing with only WS Buffer is already enough to result in plasmid pure enough for DNA sequencing and other applications. As one 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 enough to remove contaminants from a higher volume of culture. This kind of product only allows 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 MX1 Buffer. Degraded RNA does not bind well as undegraded RNA to the membrane in the presence of chaotropic salts, thus degraded RNA is washed off with wash buffer (WF 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 WF 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, an user does 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 OD260 of the plasmid eluant (degraded RNA also absorbs light at wavelength of 260 nm), thus misleading the users 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 Viogene's kit solves these problems. |
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Can PCR-M® Clean Up System be used to clean up DIG-labeled DNA fragment? |
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Yes, PX Buffer does not affect the chemically linked DIG on dNTP. Similarly, this system can be used to clean up 32P-labeled DNA fragment. |
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What are the applications of PCR-M® Clean Up System? What is the difference between this system and gel extraction system? |
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Both systems can be used to clean up DNA fragments (100-bp to 10-kb) from enzymes, salts, and dNTPs. PCR-M® Clean Up System is a cheaper choice for cleaning up DNA fragments in solution. When a specific DNA fragment is to be purified, gel electrophoresis is needed. A gel slice containing the desired DNA fragment is excised, and DNA is extracted using Gel-M® Gel Extraction System. |
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Can Mini-M® plasmid DNA extraction system be applied on Gram(+) bacteria? |
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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 MX1 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 MX2, and follow the rest of the protocol. For certain Gram(+) bacteria with thin cell wall, such as Lactobacillus, applying of double amount of MX1, MX2, and MX3 Buffer may have been enough to lyse the cells. Yet, we still recommend treating Gram(+) bacteria with lysozyme to facilitate cell lysis. |
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Can PCR-M® Clean Up System be used to clean up dye terminator from sequencing reaction? |
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It is not recommended to use this system to clean up sequencing reaction because PCR products smaller than 100-bp cannot be recovered effectively, thus making reading of the first 80-100 nucleotide sequence unfeasible. |
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Why DNA recovery from an enzymatic reaction solution is lower than expected? |
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When the pH of the enzymatic reaction solution is higher than 7.5, DNA recovery will be reduced. In this case, add 10 µl of 3M potassium acetate of pH 5.0 to the DNA solution before adding PX Buffer. |
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| 10 |
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Can total RNA be extracted from paraffin-embedded tissue? |
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It is not impossible to extract RNA from it, but it involves quite a lot difficulty and the RNA yield is very low that it is only suitable for RT-PCR. The preserved conditions of the paraffin-embedded tissue are most critical. First, if the tissue had been fixed for more than 24 hours, RNA experienced modification that cannot be used for RT-PCR anymore. Second, the older the tissue block is, the poorer the RNA yield is. Tissue blocks stored for more than 6 months are thus not suitable. Third, only short-template RT-PCR (< 400-bp) can be performed. Fourth, since only a tiny bit of tissue sample is processed, it is of course hard to disrupt the sample mechanically. In this case, Proteinase K should be used to digest the sample. However, Proteinase K is not provided in the kit. Fifth, since total RNA extracted is so little, RNA carrier (like the one used in Viral RNA Extraction System) should be added to enhance RNA recovery. Sixth, paraffin needs to be removed before the tissue is to be disrupted. Viogene Total RNA Extraction System is not tuned for processing paraffin-embedded tissues. |
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Can this system completely remove primers or dimer products from PCR reactions? |
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PCR-M® Clean Up System can only effectively (>90%) removes 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 Viogene Gel-M® Gel Extraction System. |
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Is total RNA isolated by Total RNA Extraction System free of genomic DNA? |
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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. |
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How do I increase yields of total RNA? |
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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 sample is properly handled and lysed completely, do not use more than the amount of sample suggested in the protocol.
2) Thorough celluar 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. Mechnical cell disruption techniques include grinding, homogenization with douce or rotor-stator homogenizers (polytron), vortexing, sonciation, and use of bead and freezer. Complete disruption of some tissues may require using a combination of these techniques. Rotor-stator homogenizers, alone or in conjunction with other disruption techniques, generally result in higher RNA yields than other types of homogenizer.
3) Another most frequent 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, diluting the viscous lysate with RX Buffer, 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 penetrate into it. If ddH2O still retains on the membrane, pulse centrifuge the column for a few seconds to drag it into the membrane.
5) Eluting the column twice can result in a higher RNA recovery, especially when expected RNA yield is more than 30 mg. |
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Why does the RNA obtained appear smeared and degraded? |
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Three critical steps, if not done well can cause RNA degradation. They are 1) handling and storing of samples, 2) disruption of samples, 3) 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 sample is disrupted, disruption needs to be fast and thorough. Slow disruption, e.g. placing cells or tissue in RX Buffer without any additional physical shearing, may result in RNA degradation by endogenous RNase released internally, yet still inaccessible to the protein denaturant in RX Buffer.
3) After elution of RNA with RNase-free ddH2O provided in the system, store RNA at -80°C.
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 properly cleaned gel tray and tank for electrophoresis. Adding EtBr directly into the gel can also avoid possible degradation of RNA that may occur during gel staining. |
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Can Total RNA Extraction Miniprep System be used to extract RNA from blood sample? |
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This system is not suitable for samples of liquid form such as whole blood. However, it can extract RNA from buffy coat. This system cannot be used for liquid samples because liquid in sample will dilute out RX Buffer, thus reducing its lysis and RNase inactivating ability. Since only white blood cells contain RNA, one should only use buffy coat for RNA extraction. Buffy coat for RNA extraction should be prepared from fresh blood sample. |
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Why DNA recovery is lower than expected? |
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There are several possible reasons:
1) Do not overload the column with too much DNA. Higher recovery is attained when lower amount of DNA is loaded. Split loading high amount of DNA into more than one column.
2) If ddH2O is used for elution, make sure than its pH is between 7.0 and 8.5. pH lower than 7 leads to lower elution efficiency.
3) 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 absorbed into the membrane before centrifugation.
4) Large DNA fragment is eluted less readily than small DNA fragment. When the DNA product is larger than 5-kb, use elution solution preheated to 60˚C. |
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A band smaller than the desired DNA fragment is present in the eluant. What is this smaller band? |
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The smaller band may be a single-stranded form of the PCR product. The occurrence of it could be due to that elongation of the PCR product is not complete or that PCR product is denatured during the preparation. In this case, to re-anneal the single-stranded DNA by incubating the solution at 95˚C 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. |
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Why is there genomic DNA copurified with the plasmid? |
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When genomic DNA is found in the eluant, it means that genomic DNA has been sheared during cell lysis process. After MX2 Buffer is added, ensure that mixing is done very gently to prevent genomic DNA shearing. If genomic DNA is sheared, genomic DNA fragments will get into the lysate and copurified with plasmid. |
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Why do we get eluted plasmid contaminated with RNA? |
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Make sure that RNase A is added into MX1 Buffer. Store the MX1 Buffer at 4˚C. If RNase A-added MX1 Buffer is not properly stored at 4˚C 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 added into MX1 Buffer with final concentration of 50 mg/ml. Again, store the buffer at 4˚C.
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Why is it the eluted plasmid cannot be cut by restriction enzymes properly? |
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It is possible that salt residue in buffers or ethanol residue in WS Buffer is not removed completely and thus affects the downstream reaction. In case of salt residue, wash the column twice with WS Buffer. In case of ethanol residue, after washing with WS 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.
Another reason is that plasmid is denatured. Denaturation happens if incubation in MX2 Buffer has gone for too long time. This can be visualized during electrophoresis that a band migrates faster than the supercoiled form. After MX2 Buffer is added, do NOT incubate for more than 5 minutes. |
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