Because I have bright bands in the onset of well of agarose gel. I have a doubt about contamination. I used a control cDNA to verify PCR process. I attained the right Dna fragments as I expected. And cDNA ratios are very good in my samples... like control cDNA. So as I mentioned the title, is there any problem?
-I''ll use
1-wash buffer1
2-wash buffer2
3-elution operation
4- run on agarose gel my samples again?
I have a more Pcr samples in Pcr tubes. When I meant the purify the Pcr tubes actually I meant purify my PCR samples. As you see, above in picture, There is a problem in the first 12 samples. Last two are my control. The first is ladder. And I have doubt about contamination. I want to additional purifying of my PCR tubes ( Pcr samples if there is ).
I did what I said about technique. And as you said it gave me nothing. At this time, I prepared %1 agarose gel to don't stuck my samples in onset of gel. I run my samples after purifying on agarose gel so as you can see in first 4 samples the green ones after ladder. My pcr and RT process are true because of my control samples. Visually, I failed in the rna isolation as you can see the last 4 ones in the picture. I should be much more carefull while I do rna isolation. Thank you Mr. Artur for all suggestions and taking your time :)
Have you tested your RNA samples prior to cDNA synthesis? You can measure the total amount using a spectrophotometer (it does not really measure quality, just quantity), or use a nanodrop to measure both quantity and quality. A final option would be to run some of your RNA out on a 1% gel (it isn't quantitative but you should see a nice band pattern from ribosomal RNA).
Yes I tested my Rna samples Mrs.Amy. After rna isolation prior to cDNA process. I measured my Rna samples by Nanodrop. In general, my values are ranging from 1.6 to 2.1 in OD260/280. However, my OD260/230 values are close to zero. I attained some knowledges about OD230 on internet. It must be at least OD260/280 for perfect result that I read. And my nucleus acid concentration generally are 60 ng per a ul. I could'nt see as you see any bands on Rna way.
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