Dear all,
I am working on a magnetic beads based RNA extraction project. Presently, I managed to come up with a lysis to extract RNA from blood samples. However, my PCR results show inhibitory effects. It means that my RNA is not in good quality.
Any suggestions on the chemicals that I can add into my lysis buffer to improve the lysis efficiency? I tried adding Sodium Citrate and Sodium Acetate but they arent compatible to my current lysis buffer.
Let me know your ideas.
If the DNA / RNA yield is lower than you expected for the sample, there are many factors to analyze. This is usually a lysis problem - incomplete lysis is the main cause of low yield. It can also be caused by incorrect binding conditions. Be sure to use fresh, high quality ethanol to dilute buffers or add during the coupling step. It is possible that the ethanol is of poor quality or water has entered the old stock and is not at the correct concentration. If the wash buffer is not made correctly, you may be washing the extracted DNA or RNA.
Low purity: If the extracted DNA is contaminated with protein (low 260/280 ratio), you may have started with too much sample and the protein was not completely removed or dissolved. If the DNA has a poor 260/230 ratio, the problem is usually the salt from the binder or wash buffer. Ensure that the highest quality ethanol has been used to prepare the wash buffers, and if the problem persists, give the column an additional wash.
Some samples have far more inhibitors than others. Environmental samples are particularly prone to purity problems because humic substances dissolve during extraction. Humic behaves like DNA and is difficult to remove from a silica column. For this type of sample, there are special methods for removing protein and humins before the column stage.
Degradation: Basically, in RNA extraction, degradation occurs due to improper sample storage or ineffective lysis, provided that you eluted with water without RNase. For DNA extraction, degradation is not a big problem, because for PCR, DNA can be cut and it works fine. But if you were hoping not to have too much shifted DNA, you may have used too much lysis.
Special notes on purification for PCR: Purification of PCR products is not itself a DNA extraction method, but it is a pleasant and simple method because it is simply adding a high concentration of binding salts (usually between 3-5 volumes of salt per reaction volume) and centrifuging through column. Therefore, when PCR purification kits fail, it can be particularly frustrating. The first question I ask people is, "Have you checked the gel PCR results?" Because you cannot check the PCR reaction in UV light and get accurate readings. In the 260th PCR reaction, too much ultraviolet radiation is absorbed: nucleotides, detergents, salts and primers. In my experience, failure of a PCR purification kit to work is often caused by an unsuccessful reaction. But if you know you had a strong PCR product, the best approach is to just keep your share of the flux after binding. If DNA doesn't bind, that's where it is. You can always save it and then clean it up again. And then call tech support and ask to replace the kit.
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Iron or nickel oxides. The peculiarity of this method is that small nucleic acids require higher salt concentrations for strong binding to modified magnetic particles. Therefore, the concentration of the salt can be selectively varied to release the bound nucleic acid of the desired size.
Regards, Sergey
If the DNA / RNA yield is lower than you expected for the sample, there are many factors to analyze. This is usually a lysis problem - incomplete lysis is the main cause of low yield. It can also be caused by incorrect binding conditions. Be sure to use fresh, high quality ethanol to dilute buffers or add during the coupling step. It is possible that the ethanol is of poor quality or water has entered the old stock and is not at the correct concentration. If the wash buffer is not made correctly, you may be washing the extracted DNA or RNA.
Low purity: If the extracted DNA is contaminated with protein (low 260/280 ratio), you may have started with too much sample and the protein was not completely removed or dissolved. If the DNA has a poor 260/230 ratio, the problem is usually the salt from the binder or wash buffer. Ensure that the highest quality ethanol has been used to prepare the wash buffers, and if the problem persists, give the column an additional wash.
Some samples have far more inhibitors than others. Environmental samples are particularly prone to purity problems because humic substances dissolve during extraction. Humic behaves like DNA and is difficult to remove from a silica column. For this type of sample, there are special methods for removing protein and humins before the column stage.
Degradation: Basically, in RNA extraction, degradation occurs due to improper sample storage or ineffective lysis, provided that you eluted with water without RNase. For DNA extraction, degradation is not a big problem, because for PCR, DNA can be cut and it works fine. But if you were hoping not to have too much shifted DNA, you may have used too much lysis.
Special notes on purification for PCR: Purification of PCR products is not itself a DNA extraction method, but it is a pleasant and simple method because it is simply adding a high concentration of binding salts (usually between 3-5 volumes of salt per reaction volume) and centrifuging through column. Therefore, when PCR purification kits fail, it can be particularly frustrating. The first question I ask people is, "Have you checked the gel PCR results?" Because you cannot check the PCR reaction in UV light and get accurate readings. In the 260th PCR reaction, too much ultraviolet radiation is absorbed: nucleotides, detergents, salts and primers. In my experience, failure of a PCR purification kit to work is often caused by an unsuccessful reaction. But if you know you had a strong PCR product, the best approach is to just keep your share of the flux after binding. If DNA doesn't bind, that's where it is. You can always save it and then clean it up again. And then call tech support and ask to replace the kit.
------------------------
Iron or nickel oxides. The peculiarity of this method is that small nucleic acids require higher salt concentrations for strong binding to modified magnetic particles. Therefore, the concentration of the salt can be selectively varied to release the bound nucleic acid of the desired size.
Regards, Sergey
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