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Overview: There are at least three strategies for cloning PCR products. 1) T/A cloning which is the easiest cloning method. T/A cloning takes advantage of the terminal transferase activity of Taq polymerase and other non-proofreading DNA polymerases which adds a single 3'-A overhang to each end of the PCR product. The resulting PCR product is then ligated into a linear vector with a 3´ terminal 'T' or 'U' at both ends. 2) Restriction stick end cloning. A restriction enzyme target site is introduced into each of the PCR primers. The resulting PCR product and cloning vector are digested with the restriction enzymes to generate complementary ends at the PCR product and the vector which are then ligated. 3) Blunt-end cloning. Blunt-end PCR product generated by proof-reading polymerase such as the Pfu DNA Polymerase can also be cloned into a blunt-end vector.
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Protocols
  • Blunt End Cloning PCR Products (HanCock Lab)
    http://www.cmdr.ubc.ca/bobh/methods/BLUNTENDCLONIN...
    Added: Tue May 14 2002, Hits: 7202, Reviews: 0  Write review   Cached
  • Blunting a PCR fragment DOC file (The Donelson Lab, University of Iowa)
    http://www.biochem.uiowa.edu/donelson/Database%20i...
    Added: Tue Apr 26 2005, Hits: 3154, Reviews: 0  Write review  
  • Cloning of PCR Products with Phosphorylated Primers (Hideaki Shiraishi, Kyoto University)
    For cloning DNA fragments by ligating with dephosphorylated vector DNA, the fragments should have phosphates on their 5' termini. Since phosphorylation of PCR products by T4 polynucleotide kinase is inefficient, primers should be phosphorylated prior to PCR reaction.
    http://kuchem.kyoto-u.ac.jp/seika/shiraishi/protoc...
    Added: Fri Jan 30 2004, Hits: 4170, Reviews: 0  Write review   Cached
  • Cloning PCR Product (Fermentas)
    Including PCR primer design, DNA preparation, ligation, transformation...
    http://www.fermentas.com/Profiles/Kits/PDovetailKi...
    Added: Tue May 14 2002, Hits: 14730, Reviews: 1 Read reviews  Write review   Cached
  • Cloning PCR-Generated DNA Fragments (Gerard Lazo)
    http://wheat.pw.usda.gov/~lazo/methods/lazo/pcr-cl...
    Added: Tue May 14 2002, Hits: 9421, Reviews: 0  Write review   Cached
  • Direct Cloning of Blunt-end PCR Fragments (Gerard Lazo)
    http://wheat.pw.usda.gov/~lazo/methods/lazo/met4.h...
    Added: Tue May 14 2002, Hits: 4223, Reviews: 0  Write review   Cached
  • Directional PCR Product Cloning using BpiI, Eco31I and Esp3I (Fermentas)
    A common method to clone PCR products involves digestion with restriction enzymes that recognize sites introduced into PCR primers. Its drawback is that the cleavage efficiency close to the ends of a DNA fragment is not well defined what makes appropriate digestion of a PCR product difficult. This problem is overcome in an original and efficient method "Directional PCR Product Cloning using BpiI, Eco3I and Esp3I" by using one of the three well-characterized type IIS restriction endonucleases to digest a PCR fragment leaving sticky ends of 4 bases that can be ligated with complementary DNA ends generated by more than 45 restriction enzymes.
    http://www.fermentas.com/techinfo/re/dirpcrcloning...
    Added: Mon Jan 02 2006, Hits: 1724, Reviews: 0  Write review   Cached
  • One Tube PCR Cloning Method (Dr. Chun-Ming Liu, Plant Research International)
    This method has 3 reactions (restriction, polishing and ligation) carried out in one tube using ligation buffer. Believe or not, it works very well without using expensive kits.
    http://www.hybtech.org/Liu/onetube.html
    Added: Tue May 14 2002, Hits: 5248, Reviews: 0  Write review   Cached
  • One-Tube PCR Cloning (Hideaki Shiraishi, Kyoto University)
    In the following protocol, recombinant plasmids can be generated in a single reaction tube from an intact plasmid and unpolished PCR products. Although this procedure is simple and rapid, number of colonies obtained after transformation of E. coli is usually smaller than that obtained by standard cloning techniques.
    http://kuchem.kyoto-u.ac.jp/seika/shiraishi/protoc...
    Added: Fri Jan 30 2004, Hits: 3393, Reviews: 0  Write review   Cached
  • PCR Product Cloning (Michael Blaber's Lab)
    This is a very useful guide to PCR cloning. Three strategies are described in great detail including introducing a restriction site, generation of half site and TA cloning.
    http://wine1.sb.fsu.edu/bch5425/lect24/lect24.htm
    Added: Tue May 14 2002, Hits: 7953, Reviews: 0  Write review   Cached
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