By Peter S. Silverstein, Shilpa J. Buch, R. Curtis Bird (auth.), Dr. R. Curtis Bird, Dr. Bruce F. Smith (eds.)
This quantity is designed to supply an simply available creation to middle options within the software of recent recombinant DNA know-how. the 1st chapters without delay tackle the functions of polymerase chain response to quite a few difficulties in DNA cloning which are, or were, tremendous tough utilizing extra conventional ways and applied sciences. those comprise cDNA cloning and transcript mapping, mutagenesis in addition to the cloning of very lengthy transcripts and protocols utilizing restricting quantities of overall RNA. extra chapters describe methods to subtractive cloning applied sciences in addition to novel really expert expression cloning and library screening suggestions, similar to adaptations on two-hybrid research for research of protein-protein interactions or research of protein-nucleic acid interactions.
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Extra info for Genetic Library Construction and Screening: Advanced Techniques and Applications
Subsequently, with advancing technologies, DNA mutants could be created in vitro at will (Smith 1985). However, the methods were inconvenient, because the thermolabile enzymes and single-stranded DNA templates were used often with only one round of DNA synthesis. , Unique Site Elimination based on restriction digestion at a site unique to the wild type (Deng and Nickoloff 1992),into the mutagenesis methods. M. com. H. C. ) Genetic Library Construction and Screening © Springer-Verlag Berlin Heidelberg 2002 34 MICHAEL MINGFU LING and BRIAN H ROBINSON of thermostable enzymes together with double-stranded DNA templates and improving the efficiency of mutagenesis.
By incorporating restriction enzyme sites at the ends of the amplification primers, K. W. uk. C.
Also check for vector self-ligation by including a ligation without insert. Lots of colonies on plates There might be some contamination of your reaction. Try to have a negative control transformation with no plasmid as a control for contamination. Very few colonies Majority of colonies are blue or light blue with very few white colonies . Make sure you are using Taq polymerase in your PCR reaction. Try to use fresh PCR products, because the single 3' A-overhang may be degraded over time. Also try to avoid a gel purification step of your PCR product, since it may remove the A-overhang.