When the column is clogged,how can user deal with it?

(1) Genomic DNA Mini (Blood/Culture cell) Kit : whole blood, plasma, serum, buffy coat, other body fluids, lymphocytes, bacteria, Gram-negative bacteria, Gram-positive bacteria , yeast and cultured cells.

(2) Genomic DNA Mini (Tissue) Kit : animal tissues, mouse tails, buccal swabs, paraffin-embedded tissue. (ps: It also can extract genomic DNA from blood and other body fluids)

(3) Genomic DNA Mini (Plant) Kit : plant tissue.

The column will be clogged due to:

(1) Too much tissue was used.

•  Too much tissue was used. If using more than 20 mg of tissue, separate it into multiple tubes.

(2) Sample tissue was not lysed completely

•  Add additional Proteinase K and extend the incubation time in Lysis step.

•  After 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 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.

If a sample is rich in protein contents, e.g. fish flesh, 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 LYS 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 is no problem in digesting the sample completely and passing the lysate through the column, amount of the sample to be applied can be increased gradually in the next preparations.

The key is to use fresh sample 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 makes extraction of all DNA from the sample unfeasibily. 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. A high amount of contaminants not only affects DNA binding, but also may not be washed off completely, leading to be carried 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 is no problem in digesting the sample completely and passing the lysate through the column, amount of the sample to be applied can be increased gradually in the subsequent preparations.

(1) Sample tissue was not lysed completely

•  Add additional Proteinase K and extend the incubation time in Lysis step.

(2) Column was clogged at 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 GD Column matrix.

(4) Incomplete DNA Elution

•  Elute twice to increase the DNA recovery.

(1) Residual ethanol contamination

•  After Washing step, dry GD Column with additional centrifugation at full speed for 5 minutes or incubation at 60 ℃ for 5 minutes.

(2) RNA contamination

•  Perform Optional RNA degradation Step.

(3) Protein contamination

•  Reduce the sample amount.

•  After DNA Binding Step, apply 400 ml W1 Buffer to wash GD Column and centrifuge at 13,000 rpm for 30 seconds. Proceed Washing Step with Wash Buffer.

(4) Genomic DNA was degraded

•  Use fresh sample or freeze fresh sample in liquid nitrogen immediately and store at -80 ℃ .

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 subjected to degradation. It is also not advised to keep samples in buffer or medium and stored 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 smearing at an extent proportional to the storage time.

(3) Use fresh TAE or TBE running buffer for electrophoresis, repeatedly used running buffer 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. ddH 2 O is not advised in this cause because DNA fragments in H 2 O suffer from gradual degradation through acid hydrolysis readily.

(5) If DNA is to be used frequently, elute it 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. It is because genomic DNA in paraffin-embedded tissue unavoidably suffers from degradation when sample was treated and stored for a long time. DNA in this case is not suitable for Southern blotting and restriction analysis due to the smearing. However, it is applicable for PCR.