Biomedical Engineering Reference
In-Depth Information
if both polymerases failed to give a product. If a single band
cannot be obtained after optimization, then gel purify the
correct band or if the extra bands are relatively minor contami-
nants proceed with the cloning but mini-prep extra colonies
from the transformed cells to overcome any possible lowering
of cloning effi ciency.
3. For high-throughput experiments the working stocks of oligo-
nucleotide are made in a green PCR plate to aid identifi cation
when the plate is stored in a freezer.
4. For high-throughput experiments the DpnI-treated purifi ed
PCR products are stored in a purple plate PCR to aid identifi -
cation when the plate is stored in a freezer.
5. For high-throughput experiments, we use one standard volume
of PCR product in the In-Fusion™ reaction. However, for
lower throughput experiments, variable volumes can be used
to achieve a 2:1 molar ratio of fragment to vector.
6. Smaller reaction volumes can be used; for example, we have
used reactions as small as 3.3
L. Alternatively, where a PCR
product is being cloned into two (or more) vectors that have
the same homology extensions but different selectable markers
then both/all vectors may be placed in a single reaction and
the transformed cells plated out onto two plates with the
appropriate antibiotics.
7. For lower throughput applications the liquid formulation of
In-Fusion™ may be more appropriate (In-Fusion
®
HD cloning
kit; Clontech).
8. This protocol is not the manufacturer's current protocol but is
the original protocol used in OPPF-UK for many years and as
it works effi ciently we have not changed it.
9. Picking two mini-preps/construct and following the In-Fusion™
cloning protocol outlined in the text, for 4,400 constructs our
cloning effi ciency is 95 %.
μ
10. Pipettes that can be expanded and contracted to transfer
between 24- and 96-well pitches are useful. We use 8-channel
Matrix IMPACT pipettors and 6-channel Rainin Pipet-Lite™
XLS Adjustable Spacer pipettors.
11. Pick colonies using a 200
L multichannel pipette tip; leave
the tip in the well after picking the colony. Once the plate is full
the tips can then be removed using a multichannel pipette and
discarded.
12. It is a good idea to have different strains in separate blocks as
they often grow at different rates and will thus require inducing
at different times.
13. This protocol uses Ni
2+
-NTA magnetic beads; however, protocols
that use fi lter plates and Ni
2+
-NTA agarose beads (e.g., Qiagen,
Ni-NTA Superfl ow 96 BioRobot Kit (4), 969261 or GE
μ
