To obtain the best extraction efficiency, in the chaotropic salt lysate, add Proteinase K (≥ 30 mAU/ml) and perform 60°C treatment to extract the cell-free DNA for 1 hour, and extract the genomic DNA of the cell 2 hour.

• Residual ethanol contamination following Wash Step
• Dry the IG Column with additional centrifugation at 14-16,000 x g for 5 minutes or incubate at 60ºC for 5 minutes. DNA was denatured (a smaller band appeared on gel analysis)
• Incubate the eluted DNA at 95ºC for 2 minutes, and then cool down slowly to re-anneal the denatured DNA.

Gel slice did not dissolve completely

• The Gel slice was too big. If using more than 300 mg of gel slice, separate it into multiple tubes.
• Use ≤2% agarose gel to ensure optimal dissolution efficiency and DNA yield.
• Raise the incubation temperature to 60ºC and extend the incubation time.

Incorrect DNA Elution Step

• Ensure that the Elution Buffer is completely absorbed after being added to the center of the IG Column.

Incomplete DNA Elution

• If DNA fragments are larger than 8 kb, use pre-heated Elution Buffer (60-70ºC) to improve the elution efficiency.

• Add absolute ethanol (see the bottle label for volume) to IW2 Buffer then mix by shaking for a few seconds. Check the box on the bottle. Close the bottle tightly after each use to avoid ethanol evaporation.
• Reduce the sample material.
• Following ethanol addition, break up any precipitate as much as possible prior to loading to IG Column.
• Add Elution Buffer or water is added into the CENTER of the column matrix.
• Elute twice to increase yield.

• Use fresh blood as long term storage may result in fragmentation of genomic DNA.
• Using TE (10 mM Tris-HCl, 1 mM EDTA, pH8.0) for elution is beneficial as EDTA preserves DNA for long term storage. However, EDTA will affect PCR and other sensitive downstream applications.
• If using water for elution, ensure the water pH is between 7.5 and 8.5. ddH2O should be fresh as ambient CO₂ can quickly cause acidification. DNA eluted in water should be stored at -20ºC to avoid degradation

• Perform in Column DNase I Digestion to eliminate DNA contamination.
• Harvested sample immediately stabilized/inappropriate handling of starting material.
• Avoid RNase contamination by always wear gloves & mask and treat all the equipment with RNaseOUT.
   

• Insufficient disruption and homogenization/too much starting material, try to adjust it.
• RNA still bound to the IR Column membrane, elute twice to increase the yield
• Ethanol carryover; following the wash step, dry the IR Column with additional centrifugation at 14-16,000 x g for 5 minutes.

• Harvested sample not immediately stabilized/inappropriate handling of starting material
• RNase contamination

• Residual Ethanol Contamination: Following the wash step, dry the RB Column with additional centrifugation at 14-16,000 x g for 5 minutes.

• Insufficient disruption and/or homogenization/too much starting material
• Centrifugation temperature was too low (should be 20ºC to 25ºC)

• Following the Wash Step, dry the IS column by incubate at 60ºC for 5 minutes.
• Use fresh sample, long term storage may result in fragmentation of nucletic acid
• Using TE (10 mM Tris-HCl, 1 mM EDTA, pH8.0) for elution is beneficial as EDTA preserves DNA for long term storage. However, EDTA will affect PCR and other sensitive downstream applications.
• If using water for elution, ensure the water pH is between 7.5 and 8.5. ddH2O should be fresh as ambient CO₂ can quickly cause acidification. DNA eluted in water should be stored at -20ºC to avoid degradation

• Add absolute ethanol (see the bottle label for volume) to IW2 Buffer then mix by shaking for a few seconds. Check the box on the bottle. Close the bottle tightly after each use to avoid ethanol evaporation.
• Reduce the sample material.
• Following ethanol addition, break up any precipitate as much as possible prior to loading to IV Column.
• Add Elution Buffer or water is added into the CENTER of the column matrix.
• Elute twice to increase yield.

Residual ethanol contamination.
Following the Wash Step, dry the IP column by incubate at 60ºC for 5 minutes.
 

Residual salt contamination.
Perform the Wash Step twice for salt sensitive downstream applications.

RNA contamination.
Add provided RNase A to IP1 Buffer then mix by shaking for a few seconds. Check the box on the bottle then store at 2-8ºC for up to 6 months. After adding IP2 Buffer to the sample mixture, mix gently by inverting the tube 10 times then let stand at room temperature for 2-5 minutes.
 

Genomic DNA contamination.
Do not use overgrown bacterial cultures. Use only fresh cultures as they will contain less genomic DNA than old cultures. During IP2 and IP3 Buffer addition, mix gently to prevent genomic DNA shearing.

• Add absolute ethanol (see the bottle label for volume) to Wash Buffer then mix by shaking for a few seconds. Check the box on the bottle. Close the bottle tightly after each use to avoid ethanol evaporation.
• Reduce the sample material.
• Following ethanol addition, break up any precipitate as much as possible prior to loading to IP column.
• Add Elution Buffer or water is added into the CENTER of the column matrix.
• Elute twice to increase yield.
   

Incomplete buffer preparation.
For IP100 and IP300 add provided RNase A to IP1 Buffer then mix by shaking for a few seconds. Check the box on the bottle then store at 2-8ºC for up to 6 months. If precipitates have formed in IP2 Buffer, warm in a 37ºC water bath followed by gentle shaking to dissolve. Add absolute ethanol (see the bottle label for volume) to IW2 Buffer then mix by shaking for a few seconds. Check the box on the bottle. Be sure and close the bottle tightly after each use to avoid ethanol evaporation.
 

Incomplete cell culture preparation.
We recommend using a single freshly isolated E. coli colony to inoculate into 1-10 ml of LB medium. Solid and liquid medium should contain antibiotics. Do not use overgrown bacterial cultures (≤16 hours incubated in a culture tube at 37ºC with 150-180 rpm shaking).

Culture growth medium was not removed completely.

Following centrifugation in the harvesting step, use a narrow pipette tip to ensure the supernatant is completely removed.
 

Cell pellet was not resuspended completely.
Resuspend the cell pellet completely by vortex or pipette. Continue to vortex or pipette until all traces of the cell pellet have been dissolved.
 

Incorrect DNA Elution step.
Ensure that Elution Buffer, TE or water is added into the center of the IP Column matrix and is completely absorbed. If plasmid DNA are larger than 10 kb, use pre-heated Elution Buffer, TE, or water (60~70ºC). If using water for elution, ensure the water pH is ≥8.0. ddH2 O should be fresh as ambient CO₂ can quickly cause acidification.
 

No yield of plasmid DNA.
Increase volume of low-copy number plasmid to 5-7 ml. We recommend using a single freshly isolated E. coli colony to inoculate into 1-10 ml of LB medium. Solid and liquid medium should contain antibiotics. Do not use overgrown bacterial cultures. Use fresh cultures only.