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.
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.
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.
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.
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.
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.
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.
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.
The Presto™ Mini RNA Yeast Kit.