If the Sequencing Techs say of your sample something like: "poor resolution from start" or
"loss of resolution after 220 nt",
you need to read the following description of the
problem and suggestions on how to prevent it.
DNA sequencers based on capillary electrophoresis are sensitive to the presence of
certain contaminants that were not a problem with the old gel-based machines.
These contaminants may not affect your sample in any way other than sequencing -
you may be able to PCR them, restrict them or even transfect them with no problems.
We do not know the exact nature of these contaminants, but we
how they typically
get into your samples, and we know how to remove them.
very typical example of a Loss of
Resolution (LOR) sample:
Early in the run, we see
50 or 60 nt further on,
it's obvious something is wrong:
Another 120 nt, and the
resolution is terrible -
the sequence is essentially
have strong reason to believe this is
caused by an unknown contaminant in your template DNA.
Read the section at the bottom of this page for why we know this to be
Here's what you can do to fix the problem:
Clean up your bad samples:
Here are three suggestions for removing the unknown
- Perform a phenol-chloroform extraction on your template
- Perform ammonium acetate-isopropyl precipitation on
- Pass your sample through a Sephadex G50 spin column.
Don't let the contaminant get into your samples in the
Mini-preps are the most common LOR samples, and many
observed that overloaded Qiagen preps (or similar silica preps) are at
fault. When you overload these plasmid prep kits, they leave many
impurities in the DNA. Do at least one of the following:
- Grow your minipreps for a shorter time. 12 hours is
hours is better. Yes, your yield may be reduced. The DNA will sequence
- Grow smaller bacterial cultures. Be conservative! If
says no more than 5 ml of lysate, limit yourself to 3 ml! Yes, your
yield will be reduced. It's worth it.
- I've heard rumors that certain E. coli strains are
particularly problematic - specifically the 'JM' strains (JM101, JM105,
etc). Be especially conservative when prepping plasmids from these
The Sequencing Core is working to help on our end as well:
I mentioned above that Sephadex G-50 spin columns can
the mystery contaminant. We in fact use Sephadex as part of our
sequencing protocols. Ours are optimized primarily to remove
unincorporated dye-labeled nucleotides, but we have tested some
protocol changes and alternative column sources that seem to minimize
the incidence of LOR. Improvements since February 2003 have primarily
been due to this effort.
DO NOT count on us solving
this problem! It is primarily a
template purity issue, and we cannot promise to get good sequence from
who want more information on this artifact, what causes
it and what prevents it,
you may want to continue reading.
There are four known causes for the LOR problem.
Three of the failure modes are within the Core itself, and are
We watch for them all the time. The last failure mode is very common,
and is a characteristic
of entire sets of samples. In other words, it is a sample prep problem
on the part of
The known causes of the LOR problem are:
- Air bubbles in the electrophoresis capillaries
- If a capillary is partially blocked by an air bubble
occasionally occurs during filling of the cap with electrophoresis
medium), the sample in that capillary will exhibit extremely poor
resolution (if it elutes at all).
This results in sporadic failures of
samples out of sets that otherwise gave good results. The Core will
always repeat samples exhibiting sporadic LOR.
- Plugged cuvette flow ports
- The flow paths in the instrument can become blocked, in
case an entire set of samples will fail with loss-of-resolution - every
sample, including standards.
We have not seen this problem in our lab.
- Overloaded lanes
- We have been told by other Core facilities and by the
manufacturer that excessive amounts of sample (sequencing termination
products) in a capillary can cause a loss of resolution much as we
describe. We have never seen this effect, and in fact have tried to
reproduce it with no success.
- Contaminated samples
- We have time and time again seen entire sets of samples
LOR, when the rest of the samples in that set (including standards)
look fine. Samples such as these, when re-run, always give the same
result - LOR. When the samples are cleaned up (e.g. by
phenol-chloroform extraction or ammonium acetate - isopropyl
precipitation) they produce excellent results.
We can only conclude that there is a contaminant
samples that reduces resolution in our machines.