I'm doing molecular biology work. I do RNA extraction and my RNA is contaminated with gDNA. I used phenol:chloroform method to carry out RNA extraction. After the DNase treatment i found out that my RNA was also degraded. I found out this through nanodrop reading and also the band intensity in the gel electrophoresis. The DNase treatment method i used was 1.Mix : RNA - 5ug, Buffer 10x- 5ul, DNase- 5ul, Add dH2O up to 50ul 2. Incubate in room temperature for 10 min 3. Add 1ul of EDTA (25mM) 4. Incubate 65 deg C for 10 min 5. Chill at ice for 1 min Then run gel.
My question is :
1) Can you help me with DNase treatment?
2)Is it necessary to carry out DNase treatment since we only want to amplify the gene of interest through PCR?
3) Can we proceed to do RT-PCR and PCR without doing the DNase treatment?
4) What is the drawback if we continue RT-PCR and PCR without DNase treatment? How does it affect the final result?