What should be the concentration of RNase to remove RNA from DNA? And after removal of RNA how to denature RNase from DNA sample?
For a typical prep we usually used something like 10 units of DNAse free RNAse.
To get rid of it you can either bind your DNA to a spin column an wash carefully or do a Phenol/Chloroform extraction. I usually preferred the last (although it makes more work) and Would recommend it.
Hi!
May I know what DNA are you isolating?
You can add RNAse prior to Phenol extraction, then you need not follow any specific denaturing methods automatically it would be removed.
Concentrations vary from manufacture to manufacture
generally 10-15 microliters (20mg/ml stock)
Refer the following book you get A to Z information of DNA isolation (animal/plant/bacterial). Molecular cloning : a laboratory manual / J. Sambrook, E.F. Fritsch, T. Maniatis
or surf on Google.
@ Jetty Ramadevi : I have already isolated DNA from human cell line.. and I got RNA bands with DNA now I want to remove RNA from the isolated DNA. and how much RNase requires to removal of RNA.
I am not sure whether all providers use the same definition, NEB says that their RNAse H digest 1 nmol RNA in 20 min at 37°C. I always use 10 U for an hour. This RNAse H can be inactivated by an incubation at 65°C and in this case the inactivation is really working.
I am surprised that you find so much RNA in your preparation, in most protocols the RNA will be degraded during the DNA präparation.
Generally for sample 10 - 15 microliters (20 mg/ml). You may loose some DNA if you want to treat with RNAse now. Hence it is always adviceable to treat with RNAse-A & Protinsae-K while isolating itself.
Anyway what was your sample volume, you can add 10-15 uL and incubate at 37C for 30 minutes or little longer no problem. If you want to denature you can incubate it at 80C for 30 min.
Can you upload your gel picture it may give some idea about quality, quantity of your DNA sample?
go to the following link you may get some idea.
http://www.autogen.com/assets/pdf/Application%20Guide%20No.%2005-DNA%20Cells.pdf
Look Dear prior to isolation itself you gather some information if you have doubts, now a days every thing available on web (www).
Good Luck!!!
To 40 uL sample volume I add 2 uL of 2 mg/mL RNase A and incubate @ 37C for 10 min. This is prior to an ethanol precipitation.
above info is really helpful , what the conc. to be used for bacterial DNA isolation ?
i extracted DNA from soil samples and Now I need to treat purified DNA with RNAse. the DNA volume is 70 ul . what are your suggestion without losing my DNA ?
I usually add 5 ul of RNAase for 1 hour and denature RNAase it by keeping DNA at 75 for 10 minutes that may improve your ratio if is 260/280=2.15 to 1.9.I suggest follow this .
Naveen Chaudhary Then you should work on your inactivation protocol. RNAse A survives autoclaving without a substancial loss of activity, so incubating it a 75°C is not sufficient.
See this paper for details: Article RNase A (EC 3.1.27.5)
Hi everyobody,
What could it happen if you add very high concentration of RNAse in your sample?, is it there any chance that you could remove some DNA from your samples?
An ampure beads purification after RNase treatment will clean the DNA
SO we used Qiagen DNA extraction kit from FFPE samples. The quality and quantity were pathetic. It does not mention about the starting material. total microns or curls that one should use as a starting material. It makes a huge difference. I used 1 ul RNAse/30ul of eluted DNA (TE buffer). THe 260/280 came down from 2.5 to 1.9 on Nanodrop 2000. I am not sure if RNAsae will interfere with downstream applications like targeted sequencing. Any experience???
Thanks
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