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RNase should be added in which step for gDNA isolation using phenol chloroform m


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#1 123blockhead

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Posted 08 June 2011 - 07:26 PM

Hi,

i wonder if i would like to get pure gDNA without RNA contamination, in which step should i add RNase?

The protocol that i'm using:
--> lysozyme treatment overnight
--> add SDS/proteinase k + RNase and incubate at 370C for 30 min
--> incubate at 560C for 3 hours
--> phenol/chloroform extraction x 3
--> chloroform extraction
--> sodium acetate + isopropanol

i'm still getting some rRNA contamination when view under gel.
Wonder what's wrong in my extraction steps.
Anybody could give any advice?
Thanks.

Edited by 123blockhead, 08 June 2011 - 07:28 PM.


#2 bob1

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Posted 09 June 2011 - 01:14 AM

How did you determine that it is RNA not digested genomic DNA?

You are adding RNase at the correct step.

#3 123blockhead

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Posted 09 June 2011 - 01:27 AM

How did you determine that it is RNA not digested genomic DNA?

You are adding RNase at the correct step.


The attached file is the gel pic using 1kb DNA ladder.
manage to get intact band for the gDNA however there are some smear in the lower part of the gel.

Attached Thumbnails

  • genomic DNA Mabs M18,24,93,94,115,139,145,148,152,154,156,159,172 0.5% 60V 90min 20110608.JPG


#4 Curtis

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Posted 09 June 2011 - 06:56 AM

your method is ok, maybe you can add more RNase next time, doesn't harm your gDNA conc.. I've tried it.

You can alternatively add RNse in the last step, after Iso-p step. I mean you can add the RNase in the water that you're going to add to the pelleted gDNA.

I have tried both methods, but I prefer the first method, cause I'm sure my gDNA is pure and nothing is there to interfere with Nanodrop reading.

#5 bob1

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Posted 09 June 2011 - 07:19 PM

The smear is much more likely to be digested genomic DNA. For RNA you would be more likely to see the 18s and 28s bands.

#6 123blockhead

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Posted 09 June 2011 - 07:43 PM

your method is ok, maybe you can add more RNase next time, doesn't harm your gDNA conc.. I've tried it.

You can alternatively add RNse in the last step, after Iso-p step. I mean you can add the RNase in the water that you're going to add to the pelleted gDNA.

I have tried both methods, but I prefer the first method, cause I'm sure my gDNA is pure and nothing is there to interfere with Nanodrop reading.


how to get rid of RNase if added in the last step?
when we adding sds & proteinase k together with RNase and incubate at 37oC, will the proteinase k digest the RNase even though the temperature is not optimum for proteinase k? or will SDS affect the activity of RNase?

#7 123blockhead

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Posted 09 June 2011 - 07:51 PM

The smear is much more likely to be digested genomic DNA. For RNA you would be more likely to see the 18s and 28s bands.


is it possible to be degraded rRNA? when i measure OD and concentration, the A260/A280 ratio is not very good. mostly 1.5 or 2.
i sent the gel pic to one of the sequencing company's technical support and she said the gel shows ribosomal RNA contamination.
she suggests me to go through RNase treatment and phenol chloroform extraction.
i'm trying on 2 samples now and will see how's the result.

#8 Vinod

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Posted 09 June 2011 - 10:04 PM

If you are going for cloning of isolated DNA then its better to remove RNA from DNA sample but if you working with molecular marker analysis just dissolve the pallet of isolated DNA in TE buffer already contain RNase (15ul of 10mg/ml Rnase in 100ml of TE bufer). You will get good results. Tell me on what you are working.

Hi,

i wonder if i would like to get pure gDNA without RNA contamination, in which step should i add RNase?

The protocol that i'm using:
--> lysozyme treatment overnight
--> add SDS/proteinase k + RNase and incubate at 370C for 30 min
--> incubate at 560C for 3 hours
--> phenol/chloroform extraction x 3
--> chloroform extraction
--> sodium acetate + isopropanol

i'm still getting some rRNA contamination when view under gel.
Wonder what's wrong in my extraction steps.
Anybody could give any advice?
Thanks.



#9 123blockhead

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Posted 09 June 2011 - 10:09 PM

If you are going for cloning of isolated DNA then its better to remove RNA from DNA sample but if you working with molecular marker analysis just dissolve the pallet of isolated DNA in TE buffer already contain RNase (15ul of 10mg/ml Rnase in 100ml of TE bufer). You will get good results. Tell me on what you are working.


i'm gonna send my gDNA samples out for whole genome re-sequencing

#10 Vinod

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Posted 10 June 2011 - 12:22 AM

1. Add 2.5 l of RNase to 0.5 ml of crude, DNA preparation (2.5 l of RNase = 25 g of RNase, so treatment is 50 g / ml of DNA preparation).

2. Mix gently but thoroughly and incubated at 37 C for 1 hr.

3. After 1 hr, add a mixture of 0.3 - 0.4 ml of chloroform: Isoamyl alcohol (24:1) and mix thoroughly for 15 minutes till an emulsion was formed.

4. Centrifuged the emulsion at 15000 rpm for 15 minutes at 4C.

5. Take supernatant by avoiding the whitish layer at interface.

6. Re-precipitated the DNA by adding double the quantity of absolute alcohol.

7. To pellet the DNA, centrifuge the tube for 5 minutes at 10,000 rpm.

8. Wash the pellet with 70% alcohol and dried over night.

9. Re-dissolved the DNA in 500l of TE buffer.

You will definitely get good results.



If RNA quantity is more then you can treat the sample for 11/2 hr






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