The integrity and length of genomic DNA isolated with silica column-based kits is highly dependent on the quality of starting material. When using the Monarch Genomic DNA Purification Kit (NEB #T3010) with fresh starting material, users should observe DNA Integrity Numbers (DINs) of 8.5–9.5, with peak sizes of 50 to > 60 kb, as measured on an Agilent TapeStation. If isolated gDNA samples have lower DINs and peak sizes, it can usually be attributed to sample preparation and storage conditions. Exceptions do apply; for example, buccal swabs and saliva samples consists of dead cells that have undergone apoptotic degradation of the DNA content and will typically result in lower DINs.
The following samples types require special attention:
The plasma of whole blood is extremely rich in nucleases. As long as the leukocytes in the blood remain intact, these nucleases will not damage the gDNA. When blood samples are stored at 4°C, over time, the leukocytes become increasingly unstable and the DNA becomes accessible to nucleases, which will reduce the size of purified gDNA. Therefore, fresh blood samples should not be stored at 4–8°C for longer than a week. On the other hand, same-day blood samples can be more difficult to lyse and the purity of the gDNA from such samples may be less consistent. As such, it is advisable to store fresh blood samples at 4–8°C for 2–3 days before purification. For archiving samples of whole blood, storage at -80°C is recommended. Frozen blood samples will give excellent quality gDNA but it is essential that samples are not thawed before the purification procedure. During the freezing process, ice crystals damage the leukocyte cell structures and any nucleases released during thawing can rapidly degrade the gDNA. However, if frozen samples are kept frozen during the addition of the lysis components and are then incubated immediately at 56°C, the stringent reaction conditions of the Blood Lysis Buffer will ensure the gDNA is protected and that large gDNA fragments are obtained.
Frozen tissue, previously ground to powder in liquid nitrogen, will be digested within minutes as the protein present in the sample is readily available to digestion by Proteinase K. This highly accessible form of input material will ensure that nucleases can rapidly be degraded, resulting in gDNA of high integrity. However, yields may be slightly lower when using powder as compared with tissue pieces since the intact gDNA may be more difficult to completely elute from the membrane due to the large fragment size.
Tissue pieces, whether frozen or stabilized, require a longer lysis time than tissue powder. It is best to cut tissue into very small pieces, as gDNA in large tissue pieces is prone to nuclease degradation; nucleases present in the interior of the pieces are protected from Proteinase K digestion, allowing them to shear nearby gDNA while the tissue piece slowly disintegrates. If the tissue samples are cut into small enough pieces, this effect is minimized and the yield and quality of gDNA will still be excellent.
Metabolically-active tissues, often also referred to as soft organ tissues (e.g. liver, kidney, pancreas and intestine), have high nuclease content. Isolating high quality gDNA from such tissues tends to be more challenging than with other samples. However, if samples are stabilized and cut to small pieces (or are processed as frozen tissue powder), good yields can be obtained. Generally, results are best when the input amounts are lower.
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