| LOW YIELD |
| Cells |
Frozen cell pellet was thawed and/or resuspended too abruptly |
- Thaw cell pellets slowly on ice and flick tube several times to release the pellet from the bottom of the tube. Be sure to use cold PBS for resuspension, and resuspend gently by pipetting up and down 5–10 times until a uniformly turbid cell suspension is obtained and the pellet is completely dissolved.
|
| Cell Lysis Buffer was added concurrently with enzymes |
- Add Proteinase K and RNase A to sample and mix well before adding the Cell Lysis Buffer, otherwise the high viscosity of the lysate will impede proper mixing of the enzymes.
|
| Blood |
Blood was thawed, allowing for DNase activity |
- Keep frozen blood samples frozen and add Proteinase K, RNase A and Blood Lysis Buffer directly to the frozen samples. Start lysis right away and let the samples thaw upon lysis incubation.
|
| Blood sample is too old |
- Fresh (unfrozen) whole blood should not be older than a week. Older samples will show a progressive amount of DNA degradation and loss of yield.
|
| Formation of hemoglobin precipitates |
- Digestion of whole blood samples from some animal species with high hemoglobin content (e.g. guinea pig) may lead to the accumulation of insoluble hemoglobin complexes that stain and clog the membrane, leading to reduced yield and purity. Reduce Proteinase K lysis time from 5 to 3 minutes to prevent the formation of these precipitates.
|
| Tissue |
Tissue pieces are too large |
- Cut starting material to the smallest possible pieces or grind with liquid nitrogen. In large tissue pieces, nucleases will destroy the DNA before the Proteinase K can lyse the tissue and release the DNA.
|
| Membrane is clogged with tissue fibers |
- Proteinase K digestion of fibrous tissues (e.g. muscle, heart, skin, ear clips), brain tissue and all RNAlater-stabilized tissues leads to the release of small indigestible protein fibers that often gives the lysate a turbid appearance. These fibers will block the binding sites of the silica membrane reducing yield and causing protein contamination. To remove fibers, centrifuge lysate at maximum speed for 3 minutes, as indicated in the protocol. For ear clips and brain tissue, use no more than 12–15 mg input material, otherwise the fiber removal will not be complete.
|
| Sample was not stored properly |
- Samples that are stored for long periods of time at room temperature, 4°C or -20°C will show degradation and loss of the gDNA content over time. Flash freeze tissue samples with liquid nitrogen or dry ice and store them at -80°C. Alternatively, use stabilizing reagents to protect the gDNA and enable storage for longer periods of time at 4°C or -20°C.
|
| Genomic DNA was degraded (common in DNase-rich tissues) |
- Organ tissues like pancreas, intestine, kidney and liver contain significant amounts of nucleases. They should be treated with extreme care and stored properly to prevent DNA degradation. Keep frozen and on ice during sample preparation. Refer to the protocol for the recommended amount of starting material and Proteinase K to use.
|
| Column is overloaded with DNA |
- Some organ tissues (e.g. spleen, kidney, liver) are extremely rich in genomic DNA. Attempting to process quantities larger than the recommended input amounts will result in the formation of clouds of tangled, long-fragment gDNA that cannot be eluted from the silica membrane. Reduce the amount of input material to get a higher yield.
|
| Incorrect amount of Proteinase K added |
- Most samples are digested with 10 µl Proteinase K, but for brain, kidney and ear clips, using 3 µl will provide better yields.
|
| DNA DEGREDATION |
| Tissue |
Sample was not stored properly |
- Samples that are stored for long periods of time at room temperature, 4°C or -20°C will show degradation and loss of the gDNA content over time. Shock freeze tissue samples with liquid nitrogen or dry ice and store them at -80°C. Alternatively, use stabilizing reagents such as RNAlater to protect the gDNA and enable storage for longer periods of time at 4°C or -20°C.
|
| Tissue pieces are too large |
- Cut starting material to the smallest possible pieces or grind with liquid nitrogen. In large tissue pieces, nucleases will degrade the DNA before the Proteinase K can lyse the tissue and release the DNA.
|
| High DNase content of soft organ tissue |
- Organ tissues like pancreas, intestine, kidney and liver have a very high nuclease content. They should be treated with extreme care (see ‘Sample not stored properly’ section above) to prevent DNA degradation. Keep frozen and on ice during sample preparation.
|
| Blood |
Blood sample is too old
|
- Fresh (unfrozen) whole blood should not be older than a week. Older samples will show a progressive amount of DNA degradation and loss of yield.
|
| Blood sample was thawed, allowing for DNase activity |
- Thawing frozen blood samples releases DNase, causing degradation. Keep frozen blood samples frozen and add enzymes and lysis buffer directly to the frozen samples. Start lysis right away and let the samples thaw upon lysis incubation.
|
| SALT CONTAMINATION |
| |
Guanidine salt was carried over into the eluate:
The binding buffer contains guanidine thiocyanate (GTC), which shows a very strong absorbance at 220–230 nm.
The most common way that salt is introduced into the eluate is by allowing the buffer/lysate mixture to contact the upper column area.
|
- When transferring the lysate/binding buffer mix, avoid touching the upper column area with the pipet tip; always pipet carefully onto the silica membrane.
- Avoid transferring any foam that may have been present in the lysate; foam can enter into the cap area of the silica spin column.
- Take care to close the caps gently to avoid splashing the mixture into the upper cap area.
- Do not move the samples too abruptly when transferring in and out of the centrifuge.
- If salt contamination is a concern, invert the columns a few times with gDNA Wash Buffer as indicated in the protocol.
|
| PROTEIN CONTAMINATION |
| Tissue |
Incomplete digestion of the tissue sample |
- Cut samples to the smallest possible pieces for rapid and efficient lysis. Allow the sample to remain in the lysis buffer for an extra 30 minutes to 3 hours after dissolving so that any remaining protein complexes are degraded and can be more easily removed during binding and washing.
|
| Membrane is clogged with tissue fibers |
- Proteinase K digestion of fibrous tissues (e.g. muscle, heart, skin, ear clips), brain tissue and all RNAlater-stabilized tissues lead to the release of small, indigestible protein fibers that often give the lysate a turbid appearance. These fibers will block the binding sites of the silica membrane reducing yield and causing protein contamination. To remove fibers, centrifuge the lysate at maximum speed for 3 minutes as indicated in the protocol. For ear clips and brain tissue, use no more than 12–15 mg input material, otherwise the fiber removal will not be complete.
|
| Blood |
High hemoglobin content |
- Some blood samples (e.g. horse) are rich in hemoglobin,
evidenced by their dark red color. On
occasion, these samples will still appear red after
the 5-minute lysis incubation (when in fact, they
should be green). Extend lysis time by 3–5 minutes
for best purity results.
|
| Formation of hemoglobin
precipitates |
- Digestion of whole blood samples from some
animal species with high hemoglobin content (e.g.
guinea pig) may lead to the accumulation of insoluble
hemoglobin complexes that stain and clog
the membrane, leading to reduced yield and purity.
Reduce Proteinase K lysis time from 5 to 3 minutes
to prevent the formation of these precipitates.
|
| RNA CONTAMINATION |
| Tissue |
Too much input material |
- DNA-rich tissues (e.g. soft organ tissue such as spleen, liver and kidney) will become very viscous during lysis and this may inhibit RNase A activity. Do not use more than the recommended input amount.
|
| Lysis time was insufficient |
- Tissue samples benefit from extending the lysis time by 30 minutes to 3 hours after the tissue piece has completely dissolved. Not only may a slightly higher yield be expected, additionally, the efficiency of the subsequent RNase A digestion is significantly higher.
|
| TISSUE DIGESTION TAKES TOO LONG |
| |
Tissue pieces too large |
- Cut tissue pieces to the smallest possible size or grind with liquid nitrogen before starting lysis.
|
| Tissue pieces are stuck to bottom of tube |
- Vortex to release pieces from the tube bottom. Vortex immediately after adding Proteinase K and Tissue Lysis Buffer to the tissue sample. Make sure that all tissue pieces can float freely.
|
Too much starting material
|
- Use input amount indicated in the protocol for best results.
|
| TISSUE LYSATE APPEARS TURBID |
| |
Formation of indigestible fibers |
- Proteinase K digestion of fibrous tissues (e.g.
muscle, heart, skin, ear clips), brain tissue and all
RNAlater-stabilized tissues leads to the release of
small indigestible protein fibers that often gives the
lysate a turbid appearance. These fibers will block
the binding sites of the silica membrane reducing
yield and causing protein contamination. To remove
fibers, centrifuge lysate at maximum speed for 3
minutes, as indicated in the protocol. For ear clips
and brain tissue, use no more than 12–15 mg input
material, otherwise the fiber removal will not be
complete.
|
| RATIO A260/A230 > 2.5 |
| |
Slight variations in EDTA concentration in eluates
|
- If the EDTA available in the elution buffer complexes with magnesium or calcium cations, which may be associated with the isolated genomic DNA in small amounts, this will lead to small differences in the free EDTA concentration in the eluate. At NEB, we have observed EDTA has a strong influence on the 230 nm absorbance and a minute concentration reduction of free EDTA may lead to a higher than usual A260/A230 ratio. In some cases, this ratio exceeds a value of 3.0 and is consistent with highly pure samples. In these cases, the elevated value does not have any negative effect on downstream applications
|
