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Problem cDNA synthesis/ 2 step RT


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6 replies to this topic

#1 maitai

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Posted 07 February 2010 - 09:53 AM

Hey,

I'm new to the field of RT PCR etc and I have several problems since I don't get any bands on my gel after 2nd strand PCR.

First I've isolated the RNA and checked it on a gel. Then I used Omniscript RT to synthesize 1st strand cDNA following the protocol. Here I used oligo-dt-primer to amplify all mRNA.

Then I used gene specific primers for my 2nd strand PCR, but when I checked the result on a gel I don't get any bands. The annealing temperature was adjusted to the primers tm.

My genes/cDNA are quiet long, one is 1,5kb and the other is 3,kb.

Any idea what I could change to get a result?

Thanks for your help...

#2 stylothecancer

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Posted 07 February 2010 - 08:17 PM

Hi maitai,

Have you check on the RNA integrity? And may I know what buffer do you used to dissolve your RNA?

#3 maitai

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Posted 08 February 2010 - 06:29 AM

hey,

i've checked my rna integrity with a spectrometer and my ratios were quiet fine.

well i've done rna extraction 2 times, first with "SV Total RNA Isolation System" kit of promega, there you have to elute RNA with depc treated water. the second time, i've made a TRI reagent rna extraction. my ratios in the TRI reagent extraction weren't as fine. here again i used depc treated water to dissolve the rna.

well maybe my rt isn't working or i have some problems with my gene specific primers...

any suggestion what else could be wrong?

#4 phage434

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Posted 08 February 2010 - 06:48 AM

I would adjust your PCR annealing temperature well below the Tm of your primers. Try 55 C, which almost always works with well designed primers.

#5 maitai

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Posted 08 February 2010 - 12:48 PM

I've tried it with 55 and with 52 and it doesn't work... :D

#6 phage434

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Posted 08 February 2010 - 07:40 PM

Well, the more you tell us the easier it will be to figure out the problem. What are the primers? What is the gene? What exactly are your reaction conditions (components, cycling parameters). Can you run a positive control on a different gene?

#7 stylothecancer

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Posted 08 February 2010 - 11:22 PM

hey,

i've checked my rna integrity with a spectrometer and my ratios were quiet fine.

well i've done rna extraction 2 times, first with "SV Total RNA Isolation System" kit of promega, there you have to elute RNA with depc treated water. the second time, i've made a TRI reagent rna extraction. my ratios in the TRI reagent extraction weren't as fine. here again i used depc treated water to dissolve the rna.

well maybe my rt isn't working or i have some problems with my gene specific primers...

any suggestion what else could be wrong?


Hi Maitai,

For your information, RNA integrity can't be check via spectrophotometer. Via spectrophotometer, you can only access the purity and the concentration of your RNA. RNA integrity can be check via agarose gel and Bioanalyzer.

Since you are using oligo dt; I presume the RNA you are isolating is having polyA tail. For your case, the RNA integrity is pretty important as your amplicon size is pretty large.




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