RT-PCR problems

jkreps at biochem.umass.edu jkreps at biochem.umass.edu
Thu May 4 07:12:50 EST 1995


>Dear Netters,
>
>We are having difficulties trying to amplify a 4.5 kb message from
>Arabidopsis by RT-PCR using specific primers (ie taken from published
>sequences).  Before we embark on empirically optimizing each of the numerous
>variables, does anyone out there have advice borne from experience
>attempting similar longish RT-PCRs?  Is poly A purification recommended for
>example (we are using 1 ug total RNA per RT reaction).  We can successfully
>amplify an unrelated sequence of 1.5 kb by our methods, but this falls
>somewhat short of an adequate positive control for both the RT step and the
>PCR. 
>
>Any advice greatly appreciated!
>
>David McCurdy
>
>
>
Dear David,

As a graduate student I successfully amplified a 1.8 kb mRNA from
Arabidopsis, which is not the same as 4.5 kb but I thought I would share my
experiences.  I used a kit (after trying various homemade combinations) from
BRL that had their "Superscript" modified MMLV RT.  In talking with the BRL
tech staff, I got the following info:  assuming your message is relatively
abundant, 1 ug of total RNA should be enough and priming with oligo dT is
better than using a specific primer.  The RT reaction is not thought to be
the limiting step, rather it is the long amplification of the cDNA.  Have
you tried your primers on gen. DNA to see if they will amplify?  What I had
to do was modify the amplification parameters (relative to the run of the
mill reaction)  so that there was enough elongation time, 30 sec for 1 kb,
and increase the time at denaturation up to 2  to 3 min.  The increased time
at elong. and denaturation temps meant that I also had to use more enzyme (2
to 3 U per 100 ul).  I modified the reaction conditions based on the
experiences of people around me who were amplifying >3 kb sized fragments.
I hope this is of help.


Joel A. Kreps
Postdoc, Anne Simon's Lab
Univ. of Mass. at Amherst, USA  




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