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Protocols
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Blunt-End Cloning
(The Donelson Lab, University of Iowa)
http://www.biochem.uiowa.edu/donelson/Database%20i...
Added: Tue Apr 26 2005, Hits: 7238, Reviews: 0 Write review -
Cloning from Low Melt Agarose
(Nonet Lab - Washington University School of Medicine)
A quick method for cloning fragments with the following ends: 1) a double cut fragment being cloned into a double cut vector if the two restriction sites are non-cohesive. 2) a single cut fragment being cloned into a single cut vector if the vector has been properly dephosphorylated. 3) A small blunt fragment, such as PCR product, being cloned into a dephosphorylated blunt vector.
http://thalamus.wustl.edu/nonetlab/ResourcesF/clon...
Added: Sat Nov 01 2003, Hits: 3005, Reviews: 0 Write review Cached -
Ligations in Low Melting Temperature Agarose
(Donis Keller Lab)
Ligation of plasmid vector and insert DNAs is carried out directly in melted agarose gel slices. This method is used for subcloning well defined restriction fragments and is probably not suitable for genomic library construction. The method works for both blunt-end and cohesive termini ligations.
http://humgen.wustl.edu/hdk_lab_manual/plasmid/pls...
Added: Tue May 14 2002, Hits: 4513, Reviews: 0 Write review Cached -
Subcloning Tips - very important heating step (Schimmelpenninck)
Put your insert and vector in an Eppendorff tube and then heat it for 5 minutes, then add ligation buffer and ligase
Added: Wed Sep 25 2002, Reviews: 0 Write review
Trouble Shootings and FAQs
- ligation problem - ligate a double cut fragment into a doubel cut vector
- ligation problems
- Ligation problem
- Subgenomic library construction - trouble ligating (bacterial) genomic fragments to bluescript K/S II
- Cut two closely located restriction sites
- Double digestion and ligation problem
- Problem with cloning a PCR fragment into a double-cut vector
- Double digestion and ligation problem
- Cloning problems - blunt ligation of a PCR fragment and a vector
- Difficulties with ligating digested pcr products to vectors
- Problem with ligation of a PCR product into a vector
- Problem with blunt Ligation of a PCR product into a vector
- How to clone annealed oligos into a vector
- Digestion and ligation problem
- Tips on improving blunt end ligation
- 3-fragment ligation
- How to ligate double-stranded oligos into a vector
- Frameshift after ligation- point-mutation in restriction site
- Tripple ligation - How can I stick 3 pieces together?
- How to do a In-gel ligation?
- To inactivate or not to inactivate ligase before transformation?
- How to confirm if ligation is sucessful?
- PCR of ligation products
- How to calculate insert:vector ratio?