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Vinod's Content

There have been 5 items by Vinod (Search limited from 02-June 19)


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#112788 Is one reference gene ever appropriate?

Posted by Vinod on 17 June 2011 - 04:03 AM in PCR, RT-PCR and Real-Time PCR

I am also trying to get the answer of this question? If you find please intimate me.



#112787 How to analyze data?

Posted by Vinod on 17 June 2011 - 04:01 AM in PCR, RT-PCR and Real-Time PCR

I am facing a problem with quantification of real-time PCR data using rotor gene-Q machine. I am using this technology for the first time. Please suggest me what are the preliminary steps required to initiate the work.
I've isolated RNA from plant at three different stage of heat treatment and want to compare the results of all. All the kit were bought from Qiagen. In my experiment tubulin was used as standard but when I go for auto detect threshold level the program say that at least two standards are required to find auto detect threshold level. I've complemented my analysis with all the treatment using 24 targeted gene with one standard. Could you please suggest me how should I process my data for final analysis to publish in scientific journal.



#112259 RNase should be added in which step for gDNA isolation using phenol chloroform m

Posted by Vinod on 10 June 2011 - 12:22 AM in Molecular Biology

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




#112254 RNase should be added in which step for gDNA isolation using phenol chloroform m

Posted by Vinod on 09 June 2011 - 10:04 PM in Molecular Biology

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.




#112003 DNase treatment after RNA extraction using RNeasy kit of qiagen

Posted by Vinod on 07 June 2011 - 02:58 AM in PCR, RT-PCR and Real-Time PCR

Hello everyone,

I isolated plant leaf RNA using RNeasy kit of qiagen. Kit itself assure DNA contamination free RNA but some minut quantity I recorded on my agarose gel. I've not done on-column DNase treatment but now I want to treat my samples with DNase. Can I use NEB DNase for this RNA. second RNA cleanup procedure is required after this treatment or not? How much RNA I should treat with DNase?

Thanks




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