Possible reasons for RAPD failure

[Roland Koelliker]

Dear Netters

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
good luck!

 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
almost infinite. 
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.
Good Look

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
in  RAPD 
application in many  plant species.  If  you have ever tried to  change
enzymes, dNTPs 
and primers, I  think that your problem is not  in enzymatics but in
mechanics.   Please 
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
microcentrifuge 
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
the machine.
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
pipetting 
one by one.
6) If you have no problem in above mentions, please try to use 1.25X buffer
rather than 
1X buffer.   I do not  know why, though  but, I usually get  better and
reliable results 
with 1.25X buffer.  If you have further question, please feel free contact
with me.  Good 
luck.

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
supplier.

     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 
problem.

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
    RAPDs.


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 
me.

  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
or thermocycler

	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.




Roland Koelliker	                  
Swiss Fed Inst Technol               
Plant Sciences                              
ETH-Zentrum, LFW-C47         
CH-8092 Zurich                          
Tel ++41-1-632 4890
Fax ++41-1-632 1153
e-mail: koelliker at ipw.agrl.ethz.ch