In-fusion protocol

2015-01-13

azim58 - 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
  o 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.
      @

o 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

* 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.
  o PCR holders last seen. . . I think I have one, and Felicia has one
  o 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
  o 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
  or Electroporation Protocol (example of electroporation performed after
  chemical transformation found in In-Fusion SMARTer Directional cDNA
  Library Construction Kit)

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