QPCR and PCR are common research focuses in many labs. Many of us have experienced inconsistencies
between samples or even triplicates from run to run or well to well. Did you know that evaporation may be to
blame? A small change in the volume of
buffers can alter the pH of the mix in the well, impacting the ability of the
enzyme to function efficiently. For
instance, at 74C for every 5ul evaporation loss of a 25ul reaction, the pH
changes by 1. What causes this
evaporation and how can we minimize it to result in more consistent, repeatable
experiments? Can we minimize evaporation
so much that we can even downscale our reactions?
A
standard one-piece plate with a polypropylene frame expands by up to 2mm during
thermal cycling (Figure 1). The issues
arise when the sealing film or strip caps expand and contract at different
rates, leaving a small space and opening up the well to evaporation. In addition, PCR blocks do not support PCR
plates from the side, allowing for the high temperatures of the thermal block
and heated lid to accelerate the expansion of the plates (Figure 2).
Evaporation in itself is an issue,
but a large issue also arises when uneven evaporation is considered. Generally evaporation occurs in the outside
wells (Figure 3). More evaporation in
one well over another means wells may have different pHs and thus differences
in enzyme efficiency. This becomes an
issue when comparing sample to sample or when trying to repeat experiments.
Two-piece
construction plates combat the issues of evaporation. In two-piece construction plates, the tubes
are made of polypropylene and the frame is polycarbonate. Polycarbonate is a more rigid plastic that
decreases the amount of thermal expansion, resulting in reduced evaporation and
this improved PCR performance. Two-piece
construction plates, due to the lower evaporation, allow for downscaling of PCR
reactions.
In
comparing the volume loss of a one-piece plate to a two-piece construction
plate, the researcher is losing money as each microliter of enzyme
evaporates. For instance, as seen in
Table 1, the outer wells of a one-piece construction plate lose almost 3 µl
while the inner wells lose over 1.5 µl due to evaporation. 4titude®’s Framestar™ two-piece construction plates,
on the other hand, only lost 0.57 µl per well in the outer wells and a little
over 0.5ul in the inner wells. A loss of
more than 6 times in the one-piece plates, adds up significantly in money
loss. If you were to assume the master
mix costs about an average of 10 cents per microliter, then in the One-piece
plates, a researcher loses $23.32 per each 96 well plate due to evaporation
(Table 1). In contrast, a researcher would only lose
$5.33 in 4titude®’s Framestar™ two-piece construction plates. In PCR plates, small amounts of evaporation
per well result in not only reproducibility issues, but also in money
loss. Two-piece construction plates
greatly reduce those issues.
4titude's
lower evaporation per well, a researcher can save money by reducing the
reaction reagent volumes without compromising results. Dr. Carolyn Denier in Berlin was able to
reduce Sybr green usage by 40%, saving over $30 per plate and still maintain
the consistent CTs she had in the past.
384 well users also see great results with 4titude® plates. Dr. Andreas Dahl in Berlin was able to
downscale his reactions as well due to decreased evaporation. By reducing reaction volumes, a researcher
can save significantly over time.
Overall,
4titude®’s Framestar™ plates and strip tubes have less thermal expansion for
improved sealing integrity resulting in lower sample evaporation. The lower sample evaporation allows customers
to maintain more consistent results and downscale their reactions to save
money. Due to the lower thermal expansion
of the 4titude® PCR plastics from MIDSCI, these plates are great for robotic
use in that they are more reliable for pipetting and stacking. The lower sample evaporation of 4titude’s PCR
plastics makes these plates and strip tubes great for low volume PCR. The plates also come in white well format for
higher sensitivity and even greater consistency from well to well.
Call MIDSCI today to purchase the 4titude® cost saving
products!
