The first thing to do when problems arise is to precipitate aliquots of the flowthrough, wash, and eluate with 0.7 volumes of iso-propanol to test for the presence of the desired nucleic acid. The resulting pellets, if any, should be rinsed in 70% ethanol, dissolved in an appropriate buffer (e.g. TE pH 7.5) and loaded onto an agarose gel. In this way one can obtain some information that may help to solve the problem. You may also contact The Nest Group, Inc. for technical assistance at (800) 347-6378.
Low YieldsIn depth discussion of conditions for purifying P1 constructs and other low copy number plasmids and cosmids.Insufficient Elution Use a second volume of elution buffer Insufficient Cell Lysis During Plasmid Use more lysis buffers and/or Preparation lysozyme as described in Special Conditions for Purifying P1 Constructs
Titer Too Low During Phage DNA Re-amplify the phage stock and Preparation see A Nuclease Free Procedure for minimum titer guidelines.
Sample pH Too High Adjust the pH of the sample being careful not to raise the salt concentration dramatically.
Load and/or Wash Buffer Salt Concentration Buffer has evaporated. Keep Too High lids tight and refer to N and L buffer preparation guidelines.
Load and/or Wash Buffer pH Too High Adjust the pH or prepare new buffer-see buffer preparation guidelines.
Elution Buffer Salt Concentration Too Low Prepare new buffer as described in buffer preparation guidelines.
Elution Buffer pH Too Low Adjust the pH or prepare new buffer-see buffer preparation guidelines.
High GC Content DNA Elute with a formamide containing buffer. . Refer to Buffer Preparation for guidelines.
DNA Pellet Was Lost After 70% EtOH Wash Decant the wash buffer very carefully or pipette off.
Chromosomal Contamination of a Plasmid PrepIn depth discussion of potential sources and solutions to genomic DNA contamination of plasmid DNA following Nucleobond AX purification.Lysis Not Gentle Enough Invert tubes during the lysis steps do not vortex. Refer to DNA contamination for suggestions.
Lysis Too Long Incubate the prep for 5 minutes only after the addition of S2 and S3. Do the S2 incubation on ice. Refer to DNA contamination for suggestions.
Lysozyme Used Omit lysozyme when working with gram-negative bacteria. Refer to DNA contamination for suggestions.
SDS in S2 Not Completely in Solution Can be seen as a white precipitate. Heat the bottle for a few minutes at 40C. Refer to DNA contamination for suggestions.
Floating Cellular Material Applied to Centrifuge prep for a longer Cartridge time and at a greater g force. Filter lysate or remove material with a cotton swab. Refer to DNA contamination for suggestions.
Poor Flow RateIn depth discussion of potential sources and solutions to insoluble white pellets following precipitation after Nucleobond AX purification.Extra Tubing Not Attached to Larger Tubing must be attached to the Columns AX 2000 cartridges.
Floating Cellular Material Applied to Centrifuge prep for a longer Cartridge time and at a greater g force. Filter or remove material with a cotton swab. Apply pressure with a syringe taking care not to exceed the maximum flow rates presented in General Characteristics.
Bacterial Lysate Unusually Viscous Dilute the sample with buffer N2 before loading. If carbohydrates are suspected, try precipitating them selectively as described in footnote 2 in nucleobond_appl/DNA-Proteinase K Procedure.
Insoluble PelletIn depth discussion of potential sources to insoluble white pellets following precipitation after Nucleobond AX purification.Pellet Vacuum Dried Air dry the pellet by carefully inverting the tube and letting it drain for a few minutes. Wipe out excess 70% EtOH with a cotton swab. Refer to Insoluble White Pellets for suggestions.
Over centrifugation of Precipitate In most case 5-10 minutes at top speed in a microfuge or 10-15 minutes in a fixed angle rotor at 12,000 x g is sufficient. Refer to Insoluble White Pellets for suggestions.
Salt in Pellet Iso-propanol was cold when added or a different alcohol was used. Equilibrate iso-propanol to room temperature and centrifuge immediately after its addition. Refer to Insoluble White Pellets for suggestions.
Resin in Pellet Too much pressure was applied to the cartridge. If possible, pellet the resin from the eluate by a short centrifugation before adding the iso-propanol and carefully remove the supernatant from the tube. See Insoluble White Pellets for more details.
Plasmid DNA Not SupercoiledIn depth discussion of potential sources and solutions to relaxed and linear forms in Nucleobond AX plasmid DNA preparations.Mechanical Shearing Usually size dependent, gently invert tubes during the lysis portion of the procedure. Refer to Relaxed and Linear Forms for suggestions.
E. coli Strain is endA+ Do not exceed the 5 minute incubations after the addition of S2 and S3. Do the incubations on ice. Perform cartridge washes immediately after the sample is loaded. Refer to Relaxed and Linear Forms for details.
Premature Culture Harvesting Grow cultures overnight and do not process them while still in the lag or log phases. Refer to Relaxed and Linear Forms for details.
Relaxation Complex Do not exceed the 5 minute incubations after the addition of S2 and S3. Do the incubations on ice. Perform cartridge washes immediately after the sample is loaded. Refer to Relaxed and Linear Forms for details.
RNA Contamination in DNA FractionWash Buffer Salt Concentration Too Low Prepare new buffer as described in buffer preparation guidelines.
Wash Buffer pH Too Low Adjust the pH or prepare new buffer-see buffer preparation guidelines.
Cellular DNA/RNA Contamination of Lambda DNA
Insufficient Extraction Matrix Used Use less lysate for a given column or use more extraction matrix. A good rule of thumb is to use 0.5 g of matrix per 10 ml of lysate.
pH of Lysate Too High Adjust pH of the lysate.
Salt Concentration of Lysate Too High Dilute the lysate before treating with the extraction matrix.
Smearing of Lambda or Other Viral DNA on Agarose Gels
E. coli Strain is endA+ Do not exceed the incubation times after the addition of T4 and S3. Add 200 µg/ml of proteinase K to the T4 incubation. Perform cartridge washes immediately after the sample is loaded. Refer to Relaxed and Linear Forms, section B for details.
Mechanical Shearing After DNA has been released from the viral particles in step 11 of A Nuclease Free Procedure, gently invert tubes after the addition of S3 and when dissolving the DNA at the end of the prep.
Insufficient Extraction Matrix Used Use less lysate for a given column or use more extraction matrix. A good rule of thumb is to use 0.5 g of matrix per 10 ml of lysate
Having specific problems? Please refer to the Nucleobond AX Trouble Shooting Guide.
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