RT-PCR troubleshooting

Thomas Burmeister tburmei at zedat.fu-berlin.de
Wed Jun 2 10:59:23 EST 1999


Hallo !
What kind of primer do you use for the RT reaction (oligo-dT, random
multimers, specific primer, ...)?
If you want to detect a specific mRNA, you will get the most efficient
reverse transcription with a specific primer. You should use the same (3')
primer, that you are using in the subsequent PCR.

Thomas Burmeister


In article <37545FBF.9BBD2C24 at pharmacy.uq.edu.au>,
s.roberts-thomson at PHARMACY.UQ.EDU.AU (Sarah Roberts-Thomson) wrote:

>I am trying to amplify a particular mRNA target and having little
>success, although other targets cause us no problems. I know the mRNA is
>present in the sample RNA we are using. We are using a one step RT-PCR
>kit and can show that the PCR step is working with our primers by using
>a brain cDNA library. We also know that all the components in this kit
>are okay as we can use it to amplify other targets. Does anyone have any
>suggestions? We have tried a range of magnesium conc, annealing temp etc
>etc.

-- 
Thomas Burmeister
<mailto:tbu at gmx.net>



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