with the mere use of the same pipettors. This is an indication that the problem can travel in the air of a
pipettor barrel (Barnes, 1994b).
To delay the appearance of these problems, start off right. Only use tips with filters in them, and
always use a dedicated set of pipettors for PCR setup, vs. post-PCR manipulations such as loading of gels.
If you can afford it, separate pre- and post-PCR manipulations in space, also. Occasionally, the problem
can be cured, at least temporarily, by bleach treatment of pipet barrels, combined with fresh stock solutions
for all reaction components.
DO DON'T
LA-PCR Tip #8 Use your 'GEL' pipettors for
Use separate sets of pipettors any solution that could go
for pre and post-PCR steps. into a PCR. If you can afford
Pre-PCR is setting up PCR. it, don't even run gels and/or
Post PCR is loading gels or PCR reaction cycling in the
cloning PCR products, etc. same room with, or upwind of,
rooms with PCR setup benches.
DO DON'T
LA-PCR Tip #9 Use too much proteinase K.
Use only 1 to 10 ug of proteinase K Use too much salt, since
per 100 ul of mouse cell suspension. proteinase K is inhibited
by salt around 0.3 M.
Most proteinase K is provided without any calcium, even though it is a calcium-requiring enzyme.
It is best to at least store it with some calcium, say 1 mM CaCl . Although famous for being a powerful
2
protease, proteinase K cannot work in very high salt, either! When these two items are allowed for, some
recipes need only 1/100 as much proteinase K. For instance, 1 ug of proteinase K is suffiicient to prepare
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