long pcr products
kowthar at zoo.utoronto.ca
Thu Dec 3 13:47:04 EST 1998
I would like to thank everyone who took the time to answer my questions.
A few of people have asked me to give details regarding the problem.
My primary interest is in trying to amplify and sequence the ITS-1
(internal) transcribed spacer) region of myxosporean parasites found in fish
tissues. To accomplish this goal, I have used a variety of universal
primers some of which are known to bind to the 18S rDNA of the parasites
and other universal primers which are expected to bind to the 5.8S rDNA
and 28S rDNA. The biggest problem is that only the 18S rDNA sequences
are known for these parasites.
In this attempt, I have used a combination of primers that are expected
to yield a 3kb fragment (very rough estimate). I know that these primers
work because I have used them before. I have enough DNA because my
positive controls using other primers gave me positive results. I
decreased the annealing temp and varied the number of cycles but neither
has helped in yielding a pcr product from this long region. Furthermore,
I have been informed that increasing the extension (greater than 3 min)
will not be necessary.
Many of the primers (universal) that I have used are also able to bind to
the fish DNA which is present in the samples. Currently, I am trying to
find a pair of primers which will bind only to the parasite DNA or will
bind to both the parasite and fish DNA to produce pcr products of
I have a few months within which time I must finish my thesis, therefore
I cannot employ techniques such as cloning which will be time consuming.
Once again, I thank all of you very much for your helpful suggestions. I
would certainly appreciate more suggestions.
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