DNAse Treatment Reaction Components - 50 µL reaction 10 ug of RNA x µL 10 x DNase buffer 5 µL TURBO DNase 1 µL nanopure water to bring volume up to 50 µL total The DNase can only handle reactions with an RNA concentration at most 200 ng/µL, as calculated in the reaction specified above.
How much RNA can be treated with DNase I?
· Thus you can aggressively digest RNA at 37c for 1 hour: this will completely remove all genomic contamination without running the risk of significant RNA breakdown which would or could happen using...
How much DNase do I need to dilute targetrna?
DNAse Treatment Reaction Components - 50 µL reaction 10 ug of RNA x µL 10 x DNase buffer 5 µL TURBO DNase 1 µL nanopure water to bring volume up to 50 µL total The DNase can only handle reactions with an RNA concentration at most 200 ng/µL, as calculated in the reaction specified above. 1.
How much DEPC is needed to inactivate RNase?
Final RNA concentration should be ≈ 20-50 ng/µl = 40-100 ng RNA/well 6) Incubate the tubes at 37˚C for 30 minutes 7) Immediately transfer the tubes to 75˚C for 10 min (exactly) to kill the DNAse I; put sample tubes on ice immediately after heating 8) Store the treated RNA at -80˚C (good for at least 3 years)
How much DNase I is needed to remove oligonucleotides?
During (in-column) DNAse I treatment my RNA-concentration drop e.g. from ~280 to 60 ng/µl, whereas my DNA-concentration drops from ~10 to ~2 ng/µl. The RNA is going to be sequenced.
How much DNase do I add?
Tip: As a rule of thumb for the DNase I digestion, use one unit of DNase I per 1 to 5 μg of total RNA in a 50 μl total volume incubated for 20 minutes at +25 to +37°C. After the additional DNase digestion step an additional purification of the RNA from the DNase I enzyme is mandatory.
How is DNase treated with RNA?
Heat inactivation: Probably the most common method of DNase inactivation is heat treatment, typically for 5 minutes at 75°C. Although this method appears straightforward, the divalent cations in the DNase digestion buffer can cause (chemically-induced) strand scission of RNA when heated.
How much RNA do you need for PCR?
3-5 ng RNA is considered enough. You should get at least 1ug RNA/million cells and can do RT with 100 ng RNA.
How do you dilute RNA concentration?
following the protocol for cDNA synthesis, you'll need to dilute the mRNA at 5ng/ul (1vol of RNA at 22ng/ul with 3.4vol of water will give you 4.4vol of RNA at 5ng/ul) and then use 4ul of your diluted sample in the reaction mixture (which is fixed at 20ul). ps: 1vol = 1 volume, whatever you want.
What should the 260 280 ratio be for RNA?
~2.0A 260/280 ratio of ~1.8 is generally accepted as “pure” for DNA; a ratio of ~2.0 is generally accepted as “pure” for RNA. Abnormal 260/280 ratios usually indicate that a sample is contaminated by residual phenol, guanidine, or other reagent used in the extraction protocol, in which case the ratio is normally low.
Is DNase treatment necessary?
Because con- struction of such primers is complicat- ed, the most commonly used method to avoid signals from the genomic DNA is to treat the tissue with DNase (7). Since this treatment is necessary to eliminate all DNA, it is a very crucial part of the in situ RT-PCR protocol.
What is a good RNA concentration?
Pure RNA has an A260/A280 ratio of 2.1, however values between 1.8-2.0 are considered acceptable for many protocols.
How is RNA concentration calculated?
After measuring the optical densities the concentration of RNA can be calculated as follows: [RNA] (μg/ml) = 40 x Dilution Factor x OD260. Nucleic acids absorb light at a wavelength of 260 nm.
How much concentration of RNA is required for cDNA synthesis?
For RNA to cDNA synthesis, we use 1 ug of RNA, then the final (20 ul) reaction mix is added to 80 ul of diH2O (final cDNA concentration 1:5), and our data is beautfiful for qPCR / rt-PCR. You can use 2 ug of total RNA for cDNA synthesis which is good enough for further qPCR studies.
What is a good RNA concentration ng uL?
On quality, RNA should always give a 260/280 ratio >2.0 and as such your samples could be slightly suboptimal. Ratios of <1.9 indicate a moderate degree of contamination which would be tolerated by RT-PCR but not more advanced applications such as microarray/RNA seq.
How do you dilute an RNA sample?
Dilution = 10 µl of RNA sample + 490 µl distilled water (1/50 dilution) Absorbance of diluted sample measured in a 1 ml cuvette (RNase-free): A260 = 0.23. Concentration of original RNA sample = 40 x A260 x dilution factor = 40 x 0.23 x 50.
How does Nanodrop measure RNA concentration?
0:4610:55How To Interpret Nanodrop Results For RNA - YouTubeYouTubeStart of suggested clipEnd of suggested clipAnd what you do is lift this arm up pipette the sample arm close the arm down and then it can take aMoreAnd what you do is lift this arm up pipette the sample arm close the arm down and then it can take a reading in a matter of seconds.
Popular Answers (1)
Ajay: could you kindly explain how DEPC treated water/plasticwares are enough to "get rid of DNA contamination"? It is interesting that you are using DEPC treatment for last 5 years to get rid of DNA.
All Answers (7)
Dont use DNAse in that case dear Milos. Infact if you are using properly DNAse/RNAse free plasticware and avoiding contamination, there is no need of using DNAse. We are doing the procedure since last 5 years and never used DNAse, instead use proper DEPC treated plasticware and dissove the RNA in DEPC water.
What is the smallest substrate for DNase I?
The smallest substrate for DNase I is a trinucleotide. Although DNase I is commonly perceived to cleave DNA nonspecifically, in practice it does show some sequence preference. For example, the enzyme is sensitive to the structure of the minor groove, and favors cleavage of purine-pyrimidine sequences. However, DNase I will cut at all 4 bases in heterogeneous dsDNA, and the specificity of cleavage at a given base usually does not vary more than 3-fold.#N#Ambion's Technical Service Department is frequently asked whether DNase I cleaves only dsDNA or whether it can also degrade single-stranded DNA (ssDNA) and the DNA in RNA-DNA hybrids. DNase I can cleave the latter 2 types of substrates, although its activity for these substrates is much reduced. For example, the specific activity of DNase I for ssDNA is about 500 times less than that for dsDNA (4). Activity on RNA-DNA hybrids is <1-2% of that for dsDNA (5). It is important to note, however, that DNase I is often used at concentrations much higher than may be necessary. For example, experiments at Ambion have shown that as much as 1 µg of 100-mer oligonucleotide can be reduced to <5-mers after a 15-min incubation with 2 U of Ambion DNase I (Cat. No. AM2222 ). As a result, the extent of cleavage of ssDNA and RNA:DNA hybrids will depend on the exact assay conditions.
What is the function of DNase I?
DNase I is a versatile enzyme that nonspecifically cleaves DNA to release 5'-phosphorylated di-, tri-, and oligonucleotide products (1). A powerful research tool for DNA manipulations, DNase I is used in a range of molecular biology applications. Some of its uses include:
How sensitive is RT-PCR?
RT-PCR has the ability to amplify a single molecule from a complex heterogeneous mixture; in fact, RT-PCR is so sensitive that when more than 40 cycles of PCR are performed, just about any reaction will produce a band in the "minus-RT" control reaction, indicating contaminating DNA.
Is DNase I a sticky enzyme?
DNase I is a sticky enzyme. In some microfuge tubes and 96-well plates we have measured that as much as 50% of the input DNase activity can adhere to the container walls in just 10 minutes! For best results use Ambion's non-stick RNase-free microfuge tubes (Cat #12450) for DNase I digestions.
Is DNase I a mystery?
While frequently used in the laboratory, the activity of DNase I is still a mystery to many researchers . Are the "units" of one source of DNase I the same as that of another? Are calcium and magnesium ions required for activity? Will DNase I degrade DNA in DNA:RNA hybrids? Can DNase I remove 100% of DNA contamination from RNA preparations? In this article we will try to answer some of the questions surrounding this commonly used enzyme.
Does DNase I cut at all 4 bases?
For example, the enzyme is sensitive to the structure of the minor groove, and favors cleavage of purine-pyrimidine sequences. However, DNase I will cut at all 4 bases in heterogeneous dsDNA, and the specificity of cleavage at a given base usually does not vary more than 3-fold.
Does Ca2+ bind to DNase I?
Since Ca2+ is known to bind tightly to DNase I and stabilize its active conformation, even micromolar levels of Ca2+ can act as a potent enzyme activator in the presence of Mg2+. The ionic strength of the reaction buffer is another factor that can affect DNase I activity.
How long does it take to autoclave RNase A?
Figure 1. Effect of Autoclaving on RNase Activity. Various concentrations of RNase A were added to PBS and autoclaved for 25 minutes. 1 µl of each solution was mixed with 1 ng of a 5 x 104 cpm RNA probe which was 304 bases long and incubated at 37°C for one hour. 5 µl of the reaction was assessed on a 5% acrylamide/8 M urea gel and exposed to film for 5 hours with an intensifying screen.
How are researchers trained in RNA isolation and analysis methods?
Researchers are usually trained in RNA isolation and analysis methods by one another or by technical manuals. Experimental procedures are often not questioned and quickly become dogma. Furthermore, it is difficult to find literature to document the "facts" taught by mentors and technical manuals. One of these potential myths is the use ...
How long does DEPC stay in water?
CO2 and EtOH are released as reaction by-products. DEPC has a half-life of approximately 30 minutes in water, and at a DEPC concentration of 0.1%, solutions autoclaved for 15 minutes/liter can be assumed to be DEPC-free. 3.
Does Tris make DEPC unavailable?
TRUE. Tris contains an amino group which "sops up" DEPC and makes it unavailable to inactivate RNase ( Figure 2 ). 1 M solutions of Tris, MOPS, HEPES and PBS were prepared, and 0.1% or 1% DEPC was added to each. One µg/ml RNase A was also added to each solution. The solutions were autoclaved and aliquots of each solution were mixed with a 304 base 32P-labeled RNA probe and incubated at 37°C for one hour. Probe integrity was assessed by electrophoresis and exposure to film. Tris and HEPES do indeed make DEPC unavailable to inactivate RNase at a DEPC concentration of 0.1% (recommended by most protocols). However, 1% DEPC is sufficient to overcome this effect. When 1M MOPS and PBS are treated with DEPC, the DEPC remains available to inactive RNase at both concentrations (0.1% and 1%). It would be impossible to predict the different interactions of DEPC with all molecular biology reagents. The most cautious approach for making RNase-free solutions would be to mix molecular biology grade powdered reagents up in DEPC-treated water. Alternatively, many pre-made nuclease-free solutions can be purchased from Thermo Fisher Scientific and other companies.
Can depc be used to inhibit translation?
However, it is also true that high levels of residual DE PC or DEPC by-products in a solution can inhibit some enzymatic reactions or chemically alter (carboxymethylate) RNA. It has been documented that DEPC byproducts in RNA samples can inhibit in vitro translation reactions (Winkler, unpublished results).
Does depc inhibit transcription?
The above data indicate that increasing amounts of DEPC increasingly inhibit transcription. Again, 0.1% DEPC is probably sufficient to inhibit most RNases with minimal effect on reactions. If DEPC is suspect in inhibiting reactions, high quality (MilliQÌ) or autoclaved water can probably be substituted into the reaction. Water can be tested using Ambion's RNaseAlert™ Kit or see Technical Bulletin #166, Nuclease and Protease Testing: Laboratory and Commercial Considerations, which describes an RNase testing protocol similar to that used in this study.
How long to incubate DNase I solution?
Add DNase I Solution dropwise to the cell suspension while gently swirling the tube. Incubate at room temperature for 15 minutes.
How to recover cells from a HBSS vial?
Rinse the vial with 1 mL of culture medium or buffer (e.g. HBSS or PBS) containing 10% FBS to recover any remaining cells, and transfer the medium to the new tube.
How to get rid of clumpy cells?
If cells still appear clumpy, pass the sample through a 37 - 70 µm cell strainer into a fresh conical tube. Rinse the sample tube three times with culture medium or buffer containing 2% FBS, then pass through the strainer.
How long to centrifuge 50 ml tube?
Centrifuge the 50 mL tube at 300 x g for 10 minutes at room temperature (15 - 25°C) to collect the cells.
Can DNase be used to reduce cell clumping?
Note: If performing downstream DNA or RNA extraction, DNase should not be used to reduce cell clumping.
What is DNase I?
DNase I (RNase-free) is ideal for: 1 Removal of contaminating genomic DNA from RNA samples 2 Degradation of DNA templates in transcription reactions
Why should EDTA be added to a final concentration of 5 mM?
EDTA should be added to a final concentration of 5 mM to protect RNA from being degraded during enzyme inactivation (3).
What is the name of the enzyme that cleaves DNA to release oligonucleotides?
DNase I, (RNase-free) is an endonuclease that nonspecifically cleaves DNA to release di-, tri- and oligonucleotide products with 5´-phosphorylated and 3´-hydroxylated ends (1,2). DNase I acts on single- and double-stranded DNA, chromatin and RNA:DNA hybrids.
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Is New England Biolabs ethical?
New England Biolabs (NEB) is committed to practicing ethical science – we believe it is our job as researchers to ask the important questions that when answered will help preserve our quality of life and the world that we live in. However, this research should always be done in safe and ethical manner. Learn more .
