long pcr products

Kowthar Salim kowthar at zoo.utoronto.ca
Thu Dec 3 13:47:04 EST 1998


Hi All,

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 
different sizes.

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.

Kowthar.



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