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.
Gel slice did not dissolve completely
Incorrect DNA Elution Step
Incomplete DNA Elution
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.
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.
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.
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