I plan to assay total cholesterol with "cholesterol assay kit". (Cayman, Cat.# 10007640) Instruments said to dilute the assay buffer 10X into 1X with HPLC-grade water. So, first I want to buy it, but the minimum size for sale is 2.5L. I only want it 100mL, so it's too much for me. I found Nuclease free water and DEPC-treated water (for PCR). Is it okay to use that instead of HPLC-grade water?
For RNAse free water I use Ambicon DEPC-treated water (autoclaved and filtered, packaged and autoclaved again), it has no "DEPC smell"-but see #3 below. My guess is they used water without carboxylic acid contaminates "and" titrated the DEPC for the RNAse level in the water and so used very little DEPC. That water has worked great for purifying total RNA from tissue and making cDNA from the mRNA.
From the Ambicon website under TechNotes (gels not shown here):
RNase and DEPC Treatment: Fact or Laboratory Myth?
1. Autoclaving is not effective at eliminating RNase in solution because the RNases simply renature as the solution cools.
FALSE, but... Autoclaving alone does indeed inactivate a substantial amount of RNase A (Figure 1-not shown here). Various concentrations of RNase A were added to PBS and autoclaved. Aliquots of each solution were mixed with a 304 base 32P-labeled RNA probe and incubated at 37°C for one hour, followed by electrophoresis and exposure to film. Without autoclaving, the probe begins to degrade at an RNase concentration of 100 pg/ml. Autoclaving inactivates enough of the RNase A to protect the probe from degradation up to a concentration of 1 µg/ml. Note that only a portion of the RNase is inactivated by autoclaving, otherwise the RNA probe would remain intact at any RNase concentration. Autoclaving alone may be sufficient to eliminate enough RNase for some applications. However, since neither the extent of RNase contamination nor at what RNase concentration the assay is sensitive is known, DEPC should be used as an added precaution. Also note that these experiments were only performed on RNase A and may not hold true for other RNases.
2. Autoclaving inactivates DEPC
TRUE (but see #6 below). Autoclaving does inactivate DEPC by causing hydrolysis of diethylpyrocarbonate. 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. Autoclaving DEPC-containing solutions should be of sufficient duration to rid the solution of any smell
FALSE. A faint EtOH smell may linger after autoclaving, but more commonly a sweet, fruity smell is observed. This is caused by the EtOH by-product combining with trace carboxylic acid contaminates and forming volatile esters. It does not mean that trace DEPC remains in the solution.
6. If 0.1% DEPC works well to inhibit RNase, 1% should work even better
TRUE, but... Increasing DEPC concentrations inactivate increasing amounts of RNase A contamination (Figure 3-not shown here). However, it is also true that high levels of residual DEPC 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). For this study, we tested transcription reactions for DEPC inhibition effects. Template DNA was dried to completion in a vacuum centrifuge and resuspended in 0.01%, 0.1% or 1% DEPC-treated water. Duplicate MAXIscript™ transcription reactions were assembled using 32P-UTP, and the same concentration of DEPC treated water bought up the volume. The reactions were incubated and percent incorporation was assessed via TCA precipitation. The average percent incorporation were as follows:
% DEPC in reaction % incorporation
0.01 64
0.10 59
1.00 53
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 Ambion's Technical Bulletin #166, Nuclease and Protease Testing: Laboratory and Commercial Considerations, which describes an RNase testing protocol similar to that used in this study.
Nuclease-free water would be your choice, residual DEPC (in DEPC-treated water) can still theoretically carboxymethylate Tyr and His residues in any enzymes you may be working with in your assay. As well, DEPC-treated water has been reported (by IDT) to degrade nucleic acids over time. Go with nuclease-free water; it is just as good as HPLC-grade. I have used HPLC-grade and nuclease-free water interchangeably for years. Given the chance that residual diethylpyrocarbonate could still be in DEPC water, why take a chance in allowing it to affect your assay in any way...?
Hi So-Jin
I'll suggest you to go for the water grade recommended by the instrument company..
bye
you can easily go with nuclease free water, that is the best option to go with if HPLC-grade water is not available.
Nuclease-free water should be of good quality for general purposes in molecular biology.
Agree with the above...
I have used both, but nuclease free is generally best.
DEPC is really most important for getting rid of RNAse for preparing and studying RNA. No need at all for that in your project
When things are stated as "HPLC grade", it means that there are no molecules that can interfere with UV detection (ie. absorbance below 230 nm...it's been a while, but I think it is specified on the label). This will be particularly important for assaying things like cholesterol.
It is less expensive per unit volume than nuclease-free (okay) or DEPC-treated water (which is something you don't want to use).
As far as I know both DNase free water and HPLC grade are prepared filtering water. In fact milliQ water and others are also "Dnase free" if you handle them carefully. I would never use DEPC treated water. You have many compound of degradation of DEPC after inactivation that do not interfere PCRs or RT but they can be a problem in other reactions (we work with NMR and we don't use it even when we are determining RNA structures). In any case, if you are not able to obtain water from a milliQ column and at the end you are going to buy it, 100ml of DNAse free water are more expensive than 1 or even 2.5l of HPLC grade water.
I use milliQ water all the time in both molecular and HPLC applications with success. The trick is as Miguel states, handle it carefully to prevent contamination.
agree with Helena... Whatever you use, making working aliquots to minimize chance of contamination and to make it easier to start with fresh aliquots if (when) you have concerns for contamination / reaction failures....
I would call/e-mail Cayman and ask them, they have very good tech support. If the cholesterol concentration is being determined by UV, then HPLC water may be required.
Okay, for that kit you are measuring fluorescence, you could take some "Milli Q water" and do a mock assay (using just a little of the 10X buffer) to see if your water has contaminants that fluoresce. Plastic ware, pipette tips, and tubes can also be a source of fluorescing compounds, just depends on how much cholesterol you have in your sample. Several control samples will let you see if that is a problem.
I was doing a fluorescent assay for nitrates/nitrites (using a Cayman kit) and some brands of pipette tips were contaminating my samples with fluorescing compounds. Was only a problem for the standards at the low end of the standard curve. The easy solution for the pipette tip contaminants was to rinse them with HPLC grade water before use, that removed the fluorescent compounds.
quick question...does anybody use DEPC treated water for PCR anymore ? we stop using it two centuries ago !!! lol
In our lab, we do lots of RT-PCR, and have plenty of lab-make DEPC treated water--but we use so little that I buy a 500mL bottle of RNAse-free water and aliquot it into 1.5 mL aliquots.
I was trained and certified at ROCHE PCR Academy (NJ) in 1996 in qPCR and was taught to use only Dnase-Rnase free water for PCR purposes because DEPC "interferes" with the reaction...maybe is not true anymore...
DEPC 'interferes' because it is not entirely broken down into carbon dioxide and ethanol (even after autoclaving 3 times)... some publications have disputed this; but I welcome that debate. The nose knows.
Thus, if DEPC is still there, even in part, it can still do what it does best - which is to carboxymethylate histidine and tyrosine residues (amino acids) in any proteins/enzymes present (which was its apparent purpose in the first place; for to disfigure the active domain of RNase enzymes). Being a non-discriminatory chemical, that means DEPC will also wreak the same havoc on reverse transcriptases, Taq DNA-dependent DNA polymerases, and YOU!
Appropriately (industrial-strength) filtered water removes all enzymes (including nucleases: DNases and RNases) by definition anyway; and that is why HPLC-grade and nuclease-free grade water are excellent alternatives here. Just because we used too much fire-power in the past (DEPC), doesn't mean it is blindly needed now...
[spoiler alert: joke in poor taste coming up]:
Perhaps there should be background checks on people who think that using DEPC-treated water is still a good idea ;]... It is a mutagen!
Elda Duran above, is absolutely correct in the advice received from the ROCHE PCR Academy (NJ). Integrated DNA Technologies (IDT in Coralville, Iowa) says the same.
Actually, IDT stated in the late 90s or early 2000s, that residual DEPC can degrade nucleic acids (oligonucleotides; AKA primers and probes) if they are stored in DEPC-treated water...
Gabbing about any lingering 'merits' of DEPC at present is like wondering if we should keep treating people with mercury and lead for disease.
Does anyone have anything good to say about DEPC or DEPC-treated water? I certainly don't.
... The post on DEPC on Wikipedia: (just looked it up) - the news is slightly worse than I stated:
Diethylpyrocarbonate (DEPC), also called diethyl dicarbonate (IUPAC name), diethyl oxydiformate, ethoxyformic anhydride, or pyrocarbonic acid diethyl ester, is used in the laboratory to inactivate RNase enzymes in water and on laboratory utensils. It does so by the covalent modification of histidine (most strongly), lysine, cysteine, and tyrosine residues.[1]
DEPC-treated (and therefore RNase-free) water is used in handling of RNA in the laboratory, to reduce the risk of RNA being degraded by RNases.
Water is usually treated with 0.1% v/v diethylpyrocarbonate for at least 2 hours at 37 °C and then autoclaved (at least 15 min) to inactivate traces of DEPC. Inactivation of DEPC in this manner yields CO2, H2O and ethanol. Higher concentrations of DEPC are capable of deactivating larger amounts of RNase, but remaining traces or byproducts may inhibit further biochemical reactions such as in vitro translation. Furthermore, chemical modification of RNA such as carboxymethylation is possible when traces of DEPC or its byproducts are present, resulting in impaired recovery of intact RNA even after buffer exchange (after precipitation).
So RNA is also prey of the beast as well...
Anyway -- that's my last addition here.
New topics to get to.
I hope So-Jin Kim finds her answer amidst the noise we all made here~
For RNAse free water I use Ambicon DEPC-treated water (autoclaved and filtered, packaged and autoclaved again), it has no "DEPC smell"-but see #3 below. My guess is they used water without carboxylic acid contaminates "and" titrated the DEPC for the RNAse level in the water and so used very little DEPC. That water has worked great for purifying total RNA from tissue and making cDNA from the mRNA.
From the Ambicon website under TechNotes (gels not shown here):
RNase and DEPC Treatment: Fact or Laboratory Myth?
1. Autoclaving is not effective at eliminating RNase in solution because the RNases simply renature as the solution cools.
FALSE, but... Autoclaving alone does indeed inactivate a substantial amount of RNase A (Figure 1-not shown here). Various concentrations of RNase A were added to PBS and autoclaved. Aliquots of each solution were mixed with a 304 base 32P-labeled RNA probe and incubated at 37°C for one hour, followed by electrophoresis and exposure to film. Without autoclaving, the probe begins to degrade at an RNase concentration of 100 pg/ml. Autoclaving inactivates enough of the RNase A to protect the probe from degradation up to a concentration of 1 µg/ml. Note that only a portion of the RNase is inactivated by autoclaving, otherwise the RNA probe would remain intact at any RNase concentration. Autoclaving alone may be sufficient to eliminate enough RNase for some applications. However, since neither the extent of RNase contamination nor at what RNase concentration the assay is sensitive is known, DEPC should be used as an added precaution. Also note that these experiments were only performed on RNase A and may not hold true for other RNases.
2. Autoclaving inactivates DEPC
TRUE (but see #6 below). Autoclaving does inactivate DEPC by causing hydrolysis of diethylpyrocarbonate. 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. Autoclaving DEPC-containing solutions should be of sufficient duration to rid the solution of any smell
FALSE. A faint EtOH smell may linger after autoclaving, but more commonly a sweet, fruity smell is observed. This is caused by the EtOH by-product combining with trace carboxylic acid contaminates and forming volatile esters. It does not mean that trace DEPC remains in the solution.
6. If 0.1% DEPC works well to inhibit RNase, 1% should work even better
TRUE, but... Increasing DEPC concentrations inactivate increasing amounts of RNase A contamination (Figure 3-not shown here). However, it is also true that high levels of residual DEPC 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). For this study, we tested transcription reactions for DEPC inhibition effects. Template DNA was dried to completion in a vacuum centrifuge and resuspended in 0.01%, 0.1% or 1% DEPC-treated water. Duplicate MAXIscript™ transcription reactions were assembled using 32P-UTP, and the same concentration of DEPC treated water bought up the volume. The reactions were incubated and percent incorporation was assessed via TCA precipitation. The average percent incorporation were as follows:
% DEPC in reaction % incorporation
0.01 64
0.10 59
1.00 53
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 Ambion's Technical Bulletin #166, Nuclease and Protease Testing: Laboratory and Commercial Considerations, which describes an RNase testing protocol similar to that used in this study.
i would opt for HPLC water instead of nuclease free or DEPC treated water. Since you will be assaying cholestrol, my take would be to use HPLC water instead as I beleive they would have removed most of the contaminants that may affect your assay readings in a mass spec. Nuclease free/ DEPC treated water (most do not use that anymore due to its carcinogenicity) are normally used for biological assays such as RNA, cDNA protocols.
Grace,
I agree, some of us just got off the original topic, sorry So-Jin!
Gary, your post rocked -don't worry - it's never bad to explore the perimeter!
If I can use milliQ water for NMR analysis, or should I go for HPLC grade water?
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