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RNA extraction and DNase treatment


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#1 rishaselva

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Posted 27 January 2015 - 05:06 AM

Dear all,

 

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? 

 

Please help



#2 CPRES

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Posted 27 January 2015 - 06:31 AM

1. DNase treatment is not necessary if the contamination is minimal but it also depends upon your downstream application of the RNA. Checkout some protocols quickly to see if you are doing things right.

 

2. If you are doing RT-PCR for a gene that has primers designed in the same exon or nearly-placed exons, then genomic DNA may contribute to the final result and needs to be degraded beforehand. However, if your primers are placed in exons which are separated 20kb because of a large intron in the middle, you don't have to worry about it.

 

3. In any case, to reduce the RNA degradation, make sure you are using RNAse free reagents (including H2O), and don't leave the RNA at RT or in heat block more than the protocol specifies.


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