1034638_PLS_QF_TS

1034638_PLS_QF_TS - Troubleshooting Guide Poor yields and...

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QIAfilter Plasmid Purification Handbook 11/2005 27 Troubleshooting Guide Poor yields and quality can be caused by a number of different factors. For optimal plasmid preparation conditions, particular attention should be paid to the lysis conditions as described in the protocol. In addition, adhering to our recommendations with respect to plasmid copy number, capacity of QIAGEN-tip, culture volume, and culture media will ensure consistent and optimal results. The following troubleshooting guide as well “General Considerations for Optimal Results” provided on our Web page www.qiagen.com/goto/plasmidinfo may be helpful in solving any problems that may arise. The scientists at QIAGEN Technical Service are always happy to answer any questions you may have about either the information and protocols in this handbook or molecular biology applications (see back cover for contact information). Comments and suggestions Low or no DNA yield No DNA in lysate (sample 1) a) Plasmid did not Please read ”Growth of Bacterial Cultures” on our Web propagate page www.qiagen.com/goto/plasmidinfo , and check that the conditions for optimal growth were met. b) Alkaline lysis was If cells have grown to very high densities or a larger inefficient amount of cultured medium than recommended was used, the ratio of biomass to lysis reagent is shifted. This may result in poor lysis conditions, because the volumes of Buffers P1, P2, and P3 are not sufficient for setting the plasmid DNA free efficiently. Reduce culture volume or increase volumes of Buffers P1, P2, and P3. Also insufficient mixing of lysis reagents will result in reduced yield. Mix thoroughly after addition of Buffers P1, P2, and P3 to achieve homogeneous suspensions. Use LyseBlue to visualize efficiency of mixing. c) Insufficient lysis for For low copy-plasmid preparations, doubling the volumes low-copy plasmids of lysis buffers P1, P2, and P3 may help to increase plasmid yield and quality (see page 11 and background on our Web page www.qiagen.com/goto/plasmidinfo ).
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QIAfilter Plasmid Purification Handbook 11/2005 28 Comments and suggestions d) Lysate incorrectly Check Buffer P2 for SDS precipitation resulting from low prepared storage temperatures and dissolve the SDS by warming. The bottle containing Buffer P2 should always be closed immediately after use. Lysis buffers prepared in the laboratory should be prepared according to the instructions on page 37. If necessary, prepare fresh Buffers P1, P2, and P3. DNA in flow-though fraction (sample 2) a) Column was Check the culture volume and yield against the capacity overloaded of the QIAGEN-tip, as detailed at the beginning of each protocol. Reduce the culture volume accordingly, or select a larger QIAGEN-tip if a higher yield is desired. For very low-copy-number plasmid and cosmid preps
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1034638_PLS_QF_TS - Troubleshooting Guide Poor yields and...

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