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)