In-fusion+protocol

Notes
 * Using alkaline phosphatase on a cut vector is not necessary for the In-Fusion reaction. In fact, better results can be obtained without using alkaline phosphatase.
 * Designing primers for in-fusion
 * primers should be designed almost as if you were doing a PCR from the 5'->3' end of a gene. On the 5' end the primers should go from the 5'->3' direction with part of the primer overlapping with the plasmid backbone (note that the primer must overlap with the very end of the DNA fragments and not start partially within the sequence; when designing primers around a restriction site area, it is important to design the primers so that they will overlap with the appropriate part of the restriction site so that the primer can bind to sequence exposed as the enzyme performs it's 3' exonuclease activity), and part of the primer overlapping with the fragment. On the 3' end the primers should run in the reverse orientation (3'->5') just like in a PCR, and part of the primer should overlap with the fragment while another part should overlap with the backbone. Note that the overlapping regions should be about 15-22 bp long. The In-Fusion reaction works because the fusion enzyme has exonuclease activity which "chews" away some of the nucleotides (about 15 or so) from the 3' end of one of the strands in the double strand. This allows several base pairs on the 5' end to be exposed to bind to complementary sequences.
 * Here is a quick sketch from Andrey showing how this process works.
 * [[image:sketch from Andrey about In-Fusion mechanism.png]]
 * primers could also be designed to linearize a plasmid at any location you want. These linearization primers should also contain 15 bp of homology to the desired overlapping fragment that you want to perform the In-Fusion with.

In-Fusion Molar Ratio Calculator http://bioinfo.clontech.com/infusion/molarRatio.do In-Fusion User Manual: www.clontech.com/xxclt_ibcGetAttachment.jsp?cItemId=17497 In-Fusion Protocol at a glance: www.clontech.com/xxclt_ibcGetAttachment.jsp?cItemId=17216

Felicia likes to do 1/4 the recommended size of a reaction

In-Fusion Protocol > or Electroporation Protocol (example of electroporation performed after chemical transformation found in In-Fusion SMARTer Directional cDNA Library Construction Kit)
 * Determine amount of vector needed using the In-Fusion Molar Ratio Calculator
 * Combine insert and vector in a PCR tube
 * Dry the contents of the tube. Place the tube in a 96 well tube holder. Place into speedvac concentrator while balancing with an empty PCR holder. Turn on concentrator, but not the heat for 20-30 min or until the tube is dry.
 * PCR holders last seen. . . I think I have one, and Felicia has one
 * Screwdriver needed to change rotor in speedvac last seen on shelf above speedvac
 * Add 4 uL H20 to dried tube. Let the tube sit for 3-4 min
 * Add 1 uL 5X In-Fusion HD Enzyme Premix (last seen in door of -20 C in yellow "Nikki" box)
 * Perform thermocycles. Use the thermocycler in the lab: select the In-Fuse2 protocol, hit block button to select block, proceed, choose other options such as no heated lid and 10 uL volume which is as low as the program will let you select
 * Note that the lid of the thermocycler generally used for in-fusion reactions does not need to be closed too tight. If the lid is closed too tight, then the tubes can be crushed.
 * Perform Precipitation Protocol After Ligation
 * Perform Chemical Transformation Protocol