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) 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.
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.
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.