Differential Display Reverse Transcriptase PCR (DD-RT-PCR)

David M. Berman eyeball at netcom.com
Sat Feb 19 00:41:44 EST 1994


szdhammi at chip.ucdavis.edu wrote:
: I am now following a new technique in our lab which no one
 in our lab has done before.  Specifically I am doing Differential
 Display Reverse Transcriptase PCR (DD-RT-PCR).
  There are some specific questions that I have.
  Can anyone out there in the net help me with these questions??    

: 1. What type of primers are used for the RT and the PCR reactions?
(10mer, 20mer, oligo-dT etc)

The downstream (11T's anchored with 2 other bases) primers are used
alone for RT and in combination with 10mers for PCR (Read Liang and
Pardee's Science paper)

: 2. What concentration of primers are used for both the reverse
transcriptase adne PCR reactions?  

It's in the paper

: 3. What is the optimal annealing temp. for primers during the PCR
reactions?

It's in the paper

: 4. We are working with a plant system, most or all of the literature deals with animal system: do any adjustments need to be made?
: (eg. starting conc. of RNA in the RT reaction) 

 This you will have
to titrate -- use the amount that gives you 50 to 200 bands in a lane.

: 5. WE ARE HAVING A PROBLEM WITH HIGH BACKGROUND.  MANY OF THE BANDS
ON THE GEL CORRESPONDING TO A SPECIFIC PRIMER-PAIR REACTION ALSO
APPEAR IN LANES IN WHICH REACTIONS ONLY CONTAINING A SINGLE PRIMER WAS
LOADED..

This is not background, this is how the technique works.  It is low
stringency and only requires 5 matches at the 3' end of the oligo to
amplify.  Many sequences are going to have matches to one oligo in two
nearby stretches in the proper orientation to amplify.

Good Luck.

: thanks a lot in advance
: Dhammika gunasekera

: szdhammi at bullwinkle.ucdavis./edu


: A
: A

-- 
                                             eyeball at netcom.com



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