Possible reasons for RAPD failure
koelliker at ipw.agrl.ethz.ch
Fri Aug 16 10:05:23 EST 1996
I did get so many nice suggestions how to solve my RAPD problem, that I
think it's helpful for many of us to see them all together.
Unfortunately my reactions still don't work, so if you have any further
suggestions please help.
Possible reasons for RAPD failure:
After my experience it may be caused by:
1. degradation,precipitation or imperfect mixing of your
stock DNA (most probably), test it on elpho and in various concentrations
on PCR (0.1ng - 250ng per 25ul reaction mixture)
2. substantial changes in your water quality
3. precipitation of your Mg stock after repeated freezing/thawing
4. (very probable) degradation of dNTPs (splitting off the third "P"
stops chain extension, aliquots should be used
5. your PCR machine
You say you have tried new taq, dNTP's, buffer etc, but have you tried
testing the pH of your water. We had a similar problem in our lab a few
months agoe and the cause was our distilled water being too low pH.
Other possibilities are your PCR machine..they need a regular service
or the temperatures may be inacurate, have you changed the source of your
gilson tips, microcentrifuge tubes, PCR tubes. The possibilities are
Good luck, RAPD is a great technique untill something goes wrong, and then
its a pig.
Occasionally primers "go bad", although this is not very
common. Also check your thermocycler - did someone change
your cycling program? Does the machine work well for other
people? - maybe it needs fixing.
My RAPDS stopped working once when I made new dilutions of my concentrated DNA
stocks. It turns out the tubes I was using to dilute the DNA were inhibiting
the reaction (don't know if the DNA was binding to the tube, some manufacturing
oil was in the tubes or what). Once I diluted the DNA in a pcr tube, it all
started working again. Good luck!
Have you ever tried a brnad new primer?
The problem of appear and disappear bands mayb a primer problem.
Do you know the phase of the moon, while you were doing the
amplifications that worked out? Was it raining or did the sun shine?
Just kidding! I'm working with Sugarcane Mapping using RAPD for more
than 2 years and some times that happens without any reason. What I
do is start all over again. I had more problems when I used a home
made Taq Polymerase, but even with BRL material it didint work some
perfectly. What I do is always have some standard DNA in my reaction.
I always use mix that doesnt need small amounts of reagents so the
errors are low. And cross my fingers.....
I've had similar problems with RAPDs and tried changing everything I
could think of as you've been doing. RAPDs are finiky, so I began looking
at things I could not change, but may have changed on its own...such as
the water that you use for your reactions. I've correlated my problems
with the time of year and the spring water runoff. Although I use
nanopure autoclaved water, there may have been changes occurring that
were greater than the changes I made to the water (ie. pH, ion
concentration). Even though distillation etc. would supposedly take care
of those things, if the changes were greater than the consistent level of
distilling and autoclaving that I do to the water, then 'leftover'
effects may contribute to an ultimate change in water quality.
This may not be the answer that you would like to hear, but it has
repeatedly happened to me, and in different provinces. My solution was to
wait several weeks, and try again with new water. It worked.
Alternatively, it may not be the water. Have you tested your
electrophoresis apparatus? ...used a positive control? ...tested
consistency in temperatures of the thermal cycler? ...are you in a lab
which might have power interuptions or inconsistencies in current? A
friend of mine discovered his problem was the inconsistency in current
being delivered to the thermal cycler. Try a positive control with other
primers ie. ribosomal or mitichondrial primers. They always work for me.
....that's a lot to test. Or maybe you've already tested it all.
Are you using the same template? If not, extractions vary greatly from
one plant to another, from one extraction to another. If you are using
the same template, have you tried a fresh extraction? Sometimes the
template degrades. I have also had the water cause problems such as those
you describe. The last thing I can think of is the thermal cycler...have
you checked the temperatures on the thermal cycler.
We have had the same problem with RAPDS. The solution is simple.
Boil the primer(s) for 10 minutes and snap freeze in liquid nitrogen.
Trust me, this works every time. Multiple freeze/thaws and long-term
storage, even at -20, results in the formation of primer secondary structure.
The symptoms usually appear as random RAPDS, some work some don't. Boiling
results in linearizing the primer, and then the reactions work. Good Luck.
I read your RAPD trouble in rapd at net.bio.net. I have much experience
application in many plant species. If you have ever tried to change
and primers, I think that your problem is not in enzymatics but in
check the followings whether or not;
1) The oil in the tube was highly pure?
2) The tube was nicely fit in the PCR machine? If you have purchased
tubes from different company recently, you would better check them. Tubes
not fit in
holes (slots) of the machine can not be received the real temperature from
3) If you put oil in slots of the machine, the oil was not too much? When
oil in slots
is so much, it will make air bubble at 94 degree of denaturation time.
Again, it will
make poor temperature control in the tube. You can clean it with cotton
ball, and add
just a little bit of oil to adjust proper contact a tube with a slot.
4) The DNA content was accurate (means too small or a lot)?
5) Pipetting was accurate? You would better make master mix rather than
one by one.
6) If you have no problem in above mentions, please try to use 1.25X buffer
1X buffer. I do not know why, though but, I usually get better and
with 1.25X buffer. If you have further question, please feel free contact
with me. Good
We had a similar experience. After sucessfully using RAPD PCR (to study
Epichloe endophytes and Bromus erectus grass) for more than two years we
began having trouble with our reactions last October. After much wasted
time and effort (and money) we found that our enzyme manufacturer had
re-formatted the enzyme they were selling in response to a "crack-down" by
the holder of the Taq polymerase patent. We subsequently tried two other
enzymes (from two other small companies) and had the same thing happen (for
the same reason). Unfortunatly, the enzyme sold by the patent holder
doesn't work (in our hands) for RAPD PCR. We have yet to solve our problem!
I would ask around to see whether anyone in Europe has found a dependable
enzyme (that they have bought recently and which is liscensed for PCR) that
works for RAPDs. Let me know if you find one!!!
Try increasing the concentration of your primers, test conc. of primers,
test them on a different species, buy new set of primers from the same
A couple of guesses for you;
1. Water quality can affect things a lot, and if you've started a new
batch, or you prepare your own (distilling is not good enough for
reliable PCR results) your source may itself be variable (e.g. some
farmer may have just sprayed something on the fields ...).
2. Lab conditions in summer are often hostile to reliable PCR (dust,
heat etc), especially if you lab is not air conditioned, or someone
turns it off at night while your PCR is running. You'll probably find
the conditions in your PCR tubes is wildy different from the winter
conditions, ramping times etc... Find a cooled room, or incubator to
put you PCR machine in.
We now use water that has been purified by reverse osmosis and then
ultrafiltered and autoclaved. We have also purchased some very expensive
water (for water) that is highly purified, which has worked the best.
Quite often if it becomes humid the PCR machines have trouble.
Things can go very wrong for no apparent reason. Might be your
I am working on RAPDs in trees such as Prosopis and Neem. I too
have observed this kind of a problem a couple of time in my work.
In both the instances, the causes for the lack of RAPDs all of a
sudden were seemingly different.
1. The first time we suddenly ran out of RAPDs was when apparently
our DNAs were old (more than 4 months old). At that time we
either rephenolised the samples once again or we simply
reprecipitated them in presence of sodium acetate. These
samples gave normal RAPD profiles.
2. The second instance this happened was very recently, after we
had made fresh batches of DNA. All of the new DNAs simply
failed to give RAPDs with any primer. In most cases at the end
of PCR we were getting smears and not bands, so apparently the
primers were being extended. We were at this stage quantifying
DNA by the EtBr on gel method. We diluted the DNAs to 25 ng/ul
according to concentration as determined by OD at 260 nm and
after that 25 to 200 ng of the DNA has consistently given good
Have you tried new dilutions of your template DNA? I experienced this
kind of trouble that were completely eliminated with fresh dilutions...
Have you checked the water? It has given us trouble occasionally.
I've seen primers' performance work well the first time, then deteriorate
after time and sometimes crap out entirely. What has helped for me is
take many small aliquots from the concentrated primer stock solution and
minimize freeze/thaw cycles, also thawing on ice instead of at RT.
I had similar problems once. Turned out to be the primers which apparently
degraded over just months in the freezer. New primers solved the problem for
I had the same problem whilst working on RAPD's in maize a
couple of years ago. I found the only way to repeat my results were to
remake the primers and re-prepare the DNA. I did not think that this would
be the problem because I was careful with my DNA and primers. However, once
I had remade the primers and reprep'd my DNA I was able to repeat the
result. It's not your reaction conditions unless you have changed polymerase
How do you store your primers and how old are they? They should be stored
at -20C minimum, being thawed out
as little as possible. It's a good idea to make two sets of primers to
minimize the thawin and refreezing.
I have my primers at 10 uM solutions stored at -80C and then dilute them to
2 uM working stocks which
are kept refrigerated. Also, you might want to check your water quality,
something simple which can
really fowl things up. We use RO water thats purified to remove 99% of
impurities, which is then autoclaved.
I then store my water in 1ml portions at -20C. The best solution is to
start everything from scratch, except
the primers. Make sure you keep all reagents frozen at atleast -20C. I
hope this helps some, I can't
say that I've never been in your position working with RAPDs.
Try recalibrating your pipetters. What size reaction mix are you using?
Very small volumes make pipetting error a serious problem, according to
responses on the 'net.
This has happened to everyone, and rarely does one find a definitive answer
such as bad taq, primers, etc. Primers apparently can lose potency over
time due to annealing, which has reportedly been solved by immersion of the
tube of primer in a hot water bath followed by snap-chilling. You might
inquire about this from others in the newsgroup.
I would check the pipetters (equipment and persons!) first, though.
One of the most important factors in the success of RAPDs is the
concentration and purity of the DNA. Have you change any of these, has the
original sample evaporate a little bit in the refrigerator?
You appear to be having the same problem that I have found. Basically
the primers degrade with time and probably freeze-thawing. I have read
somewhere that if you boil the primers for five minutes in a boiling
water bath then quick freeze in liquid nitrogen this somehow restores the
primers. We have tried this and so far have had one hundred percent
success with primers that for some reason stopped working. Alternatively
you can buy fresh primers which is more expensive.
Swiss Fed Inst Technol
Tel ++41-1-632 4890
Fax ++41-1-632 1153
e-mail: koelliker at ipw.agrl.ethz.ch
More information about the Methods