each dNTP has chelated a Mg, the amount
excess optimal for KlentaqLA is 2.5 mM.
For TaqLA, it's only 0.75 mM.
TaqLA mixtures were very problematic to me until I realized that they have more narrow optimal
ranges for Mg++ levels, and they prefer lower levels than does KlentaqLA. It is difficult to hit the
optimum magnesium concentration if one ignores the chelating activity of the dNTPs. The level of each
dNTP ranges from 100 to 250 uM amongst recipes. This can have a dramatic effect on the level of
magnesium that is left over to bind to a further magnesium binding site on the enzyme at the active site.
Excess dNTP over magnesium does not completely prevent DNA polymerase activity and PCR, but it does
prevent long PCR.
DO DON'T
LA-PCR Tip #4 Use betaine over 20 kb.
Include 1.3 M betaine, final, in the Please email exceptions
PCR reactions, and set the melt (with gel pictures) to me.
temperature of each cycle to 92-93 .
which is about 2-3 lower than without
betaine.
This will ensure that high GC targets amplify (Baskaran et al, 1996). Some average GC targets
(perhaps those with inverted repeats) nevertheless respond well to betaine, and it never seems to hurt. I
especially recommend even more betaine (2 M final) for the amplification of whole vectors containing the
colE1 replicon. I am currently evaluating 3 M betaine with promising results (Barnes, unpublished).
Why lower the melting temperature by 2-3? Although Baskaran et al. found that KlentaqLA is
more resistant to betaine than TaqLA, we find that the PCR reaction, presumably the enzyme, is a degree or
two more sensitive to heat in betaine. Concomitantly, we find that the DNA melts at least two degrees
lower. It may not seem like much, but every degree less melting temperature means somewhat less DNA
damage by depurination. We suspect that the relative sensitivity of Taq to betaine may become
insignificant at the lower melting temperatures (per cycle) of 90-93.
Although we don't have any cases of this, it seems reasonable to suppose that the annealing
temperature step of a PCR reaction should also be lowered by 2-3, for short primers that are at the edge of
stability. Alternatively, at the same annealing temperature, some PCR reactions become more specific with
(high) betaine, because alternate primer sites are prevented.
How does betaine work? We don't know for sure, but I imagine that it interferes with that third
hydrogen bond that GC base pairs have over AT base pairs. At 5.2 M betaine (Reese et al., 1993) this
makes all the DNA melt as easily AT regions without affecting specificity. I am often asked whether
betaine affects fidelity. We have only preliminary evidence on this point, but we have seen no effect so far.
DO DON'T
LA-PCR Tip #5 Depurinate your template
Preheat for 5 min. at 68C. by preheating for 1-10 min.
so that the 2 sec.window at 95 C. DNA polymerase
for the melting step can cannot cross an abasic site
be hit accurately at the This is the main current limit
first cycle. to target size.
Except: Genomic DNA can
benefit from this preheat.
Many recipes, even back at the step of preparation of the DNA template, subject the template to a
preheat step of 95-100 for several minutes. This is very bad for the template. The main danger is
depurination, leading to abasic sites on the template. DNA polymerases cannot cross an abasic site
(exceptions are repairasomes not used for PCR mixtures, at least not yet). Consider reducing any heat step
before the PCR to 80 or even lower, such as 68 C. In moderate salt, this will also prevent melting the
DNA. Double-stranded DNA template is much less sensitive to mispriming during reaction setup and
warmup, because how can the primers prime on double-stranded DNA?
An exception to this rule occurs when high quality genomic DNA is the template. The PCR
cannot start until the DNA template is denatured to the single-stranded form. High quality DNA is so long
(1 million base pairs) that it cannot denature efficiently in 2 seconds. It is therefore helpful for the first, and
only the first, heat step at 93-95 to be one or two minutes. The resulting DNA damage limits the length
of PCR product that is obtainable, but it allows the DNA to denature and become template.
DO DON'T
LA-PCR Tip #6 Heat more than 10 seconds
Set the length of each 95 (93 with for the melt step of each
betaine) melt step at the minimum time, cycle. Longer will depurinate
say 2 seconds; no more than 5-10 sec. the DNA template, and decrease
at temperature in the reaction. Robo- yields of targets, especially over 8 kb,
cycler needs 30-50 sec. to get this 5 sec. since known enzymes cannot cross
abasic sites.
For longer PCR, this is a variable worth optimizing for each brand of PCR machine, tubes, and the
volume of the reactions. For the Stratagene Robocycler, we set the heat step to 30 seconds, plus about 1/4
second per ul of reaction volume. The manufacturer's recommendations should be considered a good
starting point, but let the PCR reactions be their own thermometer. Using a fairly high GC target,
experiment to find the minimum, and then add 5-10 seconds for reliability.
DO DON'T
LA-PCR Tip #7 Use unfiltered tips for any
Use filtered pipet tips pre-PCR manipulations.
for setting up PCR reactions. Unfiltered tips are OK
Otherwise aerosols can contaminate only for loading gels.
your stock solutions with DNA an
'bad seed' from a previous PCR.
With beginner's luck, some PCR targets amplify well for the first week or two in the lab. With
beginner's (or expert's) carelessness, repeated amplification of the same target can lead to two kinds of
failure: (1) Target in the no-template control, and (2) an apparently huge product that photographs as an
ugly stain in the well, instead of a band down in the gel. These problems can travel from person to person
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