DNase Protocol: Difference between revisions
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==Procedure== | ==Procedure== | ||
#In order to DNase 20ug of RNA in a 100ul reaction, mix the following: | #In order to DNase 20ug of RNA in a 100ul reaction, mix the following: | ||
::* | ::*10μL 10X DNase buffer | ||
::* | ::*78μL dH<sub>2</sub>0 | ||
::* | ::*2μL DNase | ||
::* | ::*10μL RNA(1ug/μL) | ||
#Incubate at 37° C for 20-30 min. | #Incubate at 37° C for 20-30 min. | ||
#Add 0.1 volume resuspended DNase Inactivation Reagent. | #Add 0.1 volume resuspended DNase Inactivation Reagent. |
Revision as of 08:16, 24 May 2007
Overview
Protocol for DNase treatment of total RNA. This uses a DNase inactivating reagent and thus avoids the DNase heat inactivation step that makes me nervous.
Materials
- THAWED 10X DNase Buffer
- X μL reagent 2
- component A (reagent 2 is made up of multiple components)
- component B
- equipment 1
- equipment 2
Procedure
- In order to DNase 20ug of RNA in a 100ul reaction, mix the following:
- 10μL 10X DNase buffer
- 78μL dH20
- 2μL DNase
- 10μL RNA(1ug/μL)
- Incubate at 37° C for 20-30 min.
- Add 0.1 volume resuspended DNase Inactivation Reagent.
- Incubate 2 min at RT mixing every 30s.
- Centrifuge at 10,000xg for 1.5 min.
- Transfer solution to a new tube avoiding pelleted Inactivation Reagent.
Notes
- List troubleshooting tips here.
- You can also link to FAQs/tips provided by other sources such as the manufacturer or other websites.
- Anecdotal observations that might be of use to others can also be posted here.
Please sign your name to your note by adding '''*~~~~''': to the beginning of your tip.
References
Relevant papers and books
- Goldbeter A and Koshland DE Jr. An amplified sensitivity arising from covalent modification in biological systems. Proc Natl Acad Sci U S A. 1981 Nov;78(11):6840-4. DOI:10.1073/pnas.78.11.6840 |
- JACOB F and MONOD J. Genetic regulatory mechanisms in the synthesis of proteins. J Mol Biol. 1961 Jun;3:318-56. DOI:10.1016/s0022-2836(61)80072-7 |
- ISBN:0879697164
Contact
- Who has experience with this protocol?
or instead, discuss this protocol.