Re-DD-RT/PCR questions from szdhammi@chip.ucdavis.edu ()

babalola at MAIL.SAS.UPENN.EDU babalola at MAIL.SAS.UPENN.EDU
Wed Feb 16 20:10:57 EST 1994


The netter (szdhammi at chip.ucdavis.edu () asked the following questions on
Differential display:

1. What type of primers are used for the RT and the PCR reactions? (10mer,
20mer, oligo-dT etc)
2. What concentration of primers are used for both the reverse
transcriptase adne PCR reactions?
3. What is the optimal annealing temp. for primers during the PCR reactions?
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)
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..

My response follows:

1. I assume that you have familiarized yourself with Liang and Pardee's and
other descriptions of the use of this method. If not, you should.

2. Basically, the premise of this technique is that you anchor your 3'
primer with two 3'nucs - e.g., a typical 3' primer (both for RT and PCR)
will be 5'-TTTTTTTTTTTCA-3'. Therefore there are 12 possible combinations
of these kinds of primers. You can figure it out- just keep changing the
the 3' two nucs as fit. You can narrow down to four - See Liang et al NAR
21 (14) 3269, 1993.

3. For your 5' PCR primer you can dream up any random 10-mer oligo
sequence. For those of us in the animal world, we figured 20 of these in
combination with the 12 3' ones mentioned above, we should be in good shape
in our journey to cover the genome.

4. We use the 3'p at 2.5uM and the 5'p at 0.5uM. The key is that the dNTP
conc be kept to the bearest minimum. Read Liang and Pardee, Science 257,
967; 1992.

5. In regard to your problems with background, work out the following
parameters: a. the optimum amount of RNA - you do not need very much for
this assay, but do a titration. b. We use approximately 2uM dNTPs, but work
it out, c. I do my RT at 37 C with MMLv RT enzyme, 42 C is fine too. d.
destroy DNA in your RNA sample before proceding with DD RT. e. My PCR cond
is 94/42/72 C for 30/60/30 secs + 5 min extension at 72 C.

6. If you have anymore q. do'nt hesitate to ask on the net or direct to me.

Good luck.


Lanre Babalola 
Biol Dept., U of Penn
Phila, PA    




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