DNase Digestion and Reverse Transcription - (Jul/18/2007 )

HI Guy !

I have been trying to do reversetranscription after DNase digestion. I use the Oligo DT and MMuLV enzyme for RT.
The problem is I can't get any PCR expression even in the GAPDH. I don't know what wrong with me. I got a really good yield and concentration of RNA.
Are there any critical points during RT protocol which I have to concern ?

Thank you for any suggestion

KloD

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how did you purify you RT product ?
EDTA, SDS, and ethanol are inhibitors of PCR...

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Thank you for your asking Fred !
I did DNase digestion and use 25mM EDTA.

KloD

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Sorry Fred ! I may not answer your question yet for the previous reply. I did DNase digestion before RT.
I haven't do any purification after RT, I just use the RT product directly to PCR.

Thank a lot

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so i guess you have checked there was a product after RT ?
do you have gel of it ?
what are the buffer conditions of your RT ?

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Thank for your fast response Fred !

My RT condition is using the Oligo DT togather with MMmLv enzyme. The protocol is..

1. 2 microgram of RNA bring up the volume to 25 microlitre by DEPC
2. Add 1.5 microlitre of Oligo DT (Stock 100 ng/microlitre)
3. Heat 65 C for 5 min
4. Incubate RT 5 min
5. Add 10X Firststrand buffer, RNAsin, dNTP and MMuLV
6. Incubate 37 C for 90 min and 90 C 5 min for inactivation.

How can I check the product after RT. Can I check it by spec or gel Electrophoresis ?

Thank a lot !!!

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and what's in the first strand buffer?
how is the rest of procedure ?
is it affordable to do the reaction, and then load 2µg RNA and your reaction on agarose gel ? to check if reaction occured ?

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Hi Fred !

I use the first strand buffer from Stratagene. I have no idea for the composition of this buffer.
And this is all the process of RT and I use this product to do the the PCR.

Thank you

KloD

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Hi Klod!

My RT-PCR protocol is slightly different as the total reaction volume is performed in 20 ul and I use random primers. So that leads me to believe that the problem might be in your PCR reaction. What conditions do you use? How much of your RT do you use? I usually dilute my 20 ul of cDNA up to 200 ul and then use 5 ul of that per PCR reaction (total rxn vol of 50 ul). Also, what is your primer concentration? MgCl concentration in your reaction needs to be optimized for different primer pairs. Here's my recipe:

- 5 ul 10X PCR buffer
- x ul 50 mM MgCl2 (you have to figure this one out; I usually try out 1.25 mM up to 2 mM with 0.25 mM increments)
- 1 ul 10 mM dNTP
- 1 ul 10 uM primer 1
- 1 ul 10 uM primer 2
- 0.4 ul Taq DNA polymerase
- 5 ul cDNA (from the diluted stock)
- water up to 50 ul

I usually do 30 cycles and sometimes the temperature needs to be optimized. Just keep in mind that conditions are not the same for RT-PCR and PCR on DNA. Good luck!

Irena

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Hi Irena

Thank you very much for your suggestion

Klod

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