PlasSubclTroubleGuide

PlasSubclTroubleGuide - Plasmid Subcloning Troubleshooting...

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eZcloneSystems , 6240 Carlson Dr., Suite 1000, New Orleans, LA 70122, Tel 504-286-1759 FAX 504-286-1765 Web Site: www.ezclonesystems.com Plasmid Subcloning Troubleshooting Guide eZcloneSystems We Make Your Genes Fit! (Largest Selection of Cloning Adaptors Available Anywhere)
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eZcloneSystems , 6240 Carlson Dr., Suite 1000, New Orleans, LA 70122, Tel 504-286-1759 FAX 504-286-1765 Web Site: www.ezclonesystems.com Few or no colonies on control or test plate: 1) Some procedural mistake in processing during one or more cloning steps. This is probably not likely unless investigator is relatively inexperienced. Solution. Study procedure again and repeat cloning. 2) Cells have lost competency. Solution. If this is suspected, test competency by transfecting 0.01ng of supercoiled plasmid (should yield at least 5 colonies). If efficiency is low, make new competent cells. 3) Low fragment concentrations. A. Low Melting Point In-Gel Ligation Protocol. - Gel slices are too big. This results in a low concentration of DNA in agarose leading to low colony numbers. Solution. Repeat digest, run samples on Low Melting Point Agarose gel. Cut a thinner slice. After cutting, lay slice on its side and quickly trim excess agarose that doesn't contain DNA. Your slices should be between 30-90 ul. B. Standard fragment purification procedure. - Loss of material during purification. Run small amount of purified fragment on gel to determine whether significant loss occurred during purification. Solution. Digest, run gel, and purify fragments again. Alternatively, use "Low Melt In Gel Ligation Protocol" [can be downloaded on "Protocols" page of our web site (www.ezclonesystems.com)]. 4) Using XhoI adaptors without adding T4 polynucleotide kinase. Adding 1ul of T4 polynucleotide kinase results in approximately 100 fold higher ligation efficiency with any XhoI adaptor. This may also be moderately helpful for some other adaptors if low colony numbers are obtained. Solution. Add 1ul of T4 polynucleotide kinase to standard ligation reaction. Note: We have not tested whether T4 polynucleotide kinase works in quick ligation buffers so we recommend using standard ligation conditions for these experiments and ligate overnight. 5) Using two adaptors in a single ligation without adding T4 polynucleotide kinase.
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This note was uploaded on 11/11/2011 for the course BIO 7.012 taught by Professor Lander during the Fall '10 term at MIT.

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PlasSubclTroubleGuide - Plasmid Subcloning Troubleshooting...

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