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The following tables contain the compounds tested and indicate the compatibility of the compound tested with RNase A activity.
Experimental procedure: 45 µl of each listed reagent was added to an RNaseAlert® tube containing lyophilized substrate plus 5 µl of 10X RNaseAlert® Reaction Buffer. These mixtures were incubated for 1 hr at 37°C and then inspected for fluorescence on a UV transilluminator. Sample fluorescence was marked "-" in the "Before Addition of RNase A" if there was no visible glow and "+" if there was visible green glow.
After UV inspection and scoring, the "-" samples were further tested by adding 100 pg of RNase A and incubating an additional hour at 37°C. These tubes were then scored as above and results listed in the "After Addition of RNase A" column. RNase A was not added to enzyme solutions possessing RNase activity.
Reagents that are compatible with the RNaseAlert® technology are the ones marked "-" in the "Before Addition of RNase A" column and "+" in the "After Addition of RNase A" column.
This is not a comprehensive list of compatible reagents. It serves to illustrate some typical data using the RNaseAlert® Technology. More sophisticated monitoring can be performed with the RNaseAlert® QC System or RNaseAlert® QC System v2 using a fluorometer. Note that tips and solid surfaces are frequently positive for RNases if gloveless hands have simply touched them.
Ambion Kit Components Tested with the RNaseAlert® Technology | Before Addition of RNase A* | After Addition of RNase A* | Comments |
---|---|---|---|
DNaseZap™ Solution 1 | - | + | |
Gel Loading Buffer II | - | - | Not recommended. The dye in the loading buffer quenches RNaseAlert® fluorescence. |
MAXIscript™ Transcription Buffer, 1X | - | + | |
MEGAscript™ Reaction Buffer, 1X | - | + | |
NorthernMax™ Transfer Buffer | - | + | |
NorthernMax™-Gly Gel Loading Buffer | - | - | Inhibits RNase A activity. |
NorthernMax™-Gly Gel Running Buffer, 1X | - | + | |
Random Decamers, 10X | - | + | |
Salmon Sperm DNA (sheared) | - | + | |
THE RNA Storage Solution | - | + |
*Reagents that are compatible with RNaseAlert® technology are the ones marked "-" in the "Before Addition of RNase A" column and "+" in the "After Addition of RNase A" column.
Chemicals and Reagents Tested with the RNaseAlert® Technology | Before Addition of RNase A* | After Addition of RNase A* | Comments |
---|---|---|---|
Acetone 10% | - | + | |
Acrylamide/Bis-acrylamide 19:1, 4% | - | + | |
Acrylic acid | - | - | Inhibits RNase A activity. |
Agarose Gel Loading Buffer, 10% | - | + | |
Ammonium acetate, 500 mM | - | - | Inhibits RNase A activity. |
Ammonium persulfate, 1% | - | - | Inhibits RNase A activity. |
ATP, 1 mM | - | + | |
Betaine, 500 mM | - | + | |
Bromophenol Blue, 0.08% | - | - | Not recommended. Bromophenol blue quenches RNaseAlert® fluorescence. |
Cesium Chloride | - | - | Inhibits RNase A activity. |
Chloroform | - | - | |
Coommassie Destain | - | - | Inhibits RNase A activity. |
DEPC-treated water | - | + | |
DMSO 10% | - | + | |
EDTA 100-500 mM | - | + | 500 mM inhibits RNase A activity. |
Ethanol, 10% | - | + | |
Ethidium bromide | - | + | Not recommended. Ethidium bromide has orange/red fluorescence that makes reading the fluorescence of the RNaseAlert® substrate difficult. |
Glycerol | - | + | |
Glycerol, 10% | - | + | |
Glycine | - | + | |
GTP, 7.5 mM | - | + | |
Guanidinium hydrochloride, 100-400 mM | - | + | 400 mM inhibits RNase A activity. |
Guanidinium thiocyanate, 100-400 mM | - | + | 200 mM inhibits 5 pg of RNase A |
Magnesium acetate, 200 mM | - | - | Inhibits RNase A activity. |
Magnesium acetate, 30-100 mM | - | + | |
Magnesium chloride, 100 mM | - | + | |
Mineral Oil | - | + | |
MOPS Gel Running Buffer, 10X | - | + | |
NorthernMax Buffer (10X) | - | - | Inhibits RNase A activity. |
NP40, 1% | - | + | |
Nuclease-free water (not DEPC-treated) | - | + | |
PBS, 0.1X | - | + | |
PBS, 1X | - | + | |
PEG 8000, 4% | - | + | |
Phenol | - | - | Incubation with RNase A yielded slight violet fluoresecence. |
Potassium acetate, 100 mM, pH 8.0 | - | + | |
Potassium chloride, 200 mM | - | - | Inhibits RNase A activity. |
Potassium phosphate, 100 mM | - | - | Inhibits RNase A activity. |
RNAlater™ | - | - | |
SDS, 1% | - | + | |
Sodium Acetate, 300 mM, pH 5.2 | - | - | Inhibits RNase A activity. |
Sodium Chloride, 10-200 mM | - | + | |
Sodium Chloride, 500 mM | - | - | Inhibits RNase A activity. |
Sodium Deoxycholate, 0.4% | - | + | |
Sodium phosphate, 2 mM | - | + | |
TBE, 1X | - | + | |
TE, pH 8 | - | + | |
Tris, 100 mM, pH 8.0 | - | + | |
Tris, 100 mM, pH 7.0 | - | + | |
Tween, 1.0% | - | + | |
Urea polyacrylamide gel solution, 6% | - | + | |
Yeast Lysis Buffer | - | + | |
Zinc acetate, 100 mM | - | - | Inhibits RNase A activity. |
*Reagents that are compatible with the RNaseAlert® technology are the ones marked "-" in the "Before Addition of RNase A" column and "+" in the "After Addition of RNase A" column.
Materials Tested with the RNaseAlert® Technology | Before Addition of RNase A* | After Addition of RNase A* | Comments |
---|---|---|---|
1 week old coffee with mold | + | ND* | Glowed like the moon. |
Agilent Bioanalyzer electrodes, touched with bare hands | + | ND | Positive. |
Frank's bare hands ** | + | ND | No RNase activity detected. |
Gary's bare hands** | + | ND | Slight glow. |
George's bare hands | + | ND | Glowed brightly. |
LB with bacterial funk, contaminated | + | ND | Glowed like a Texas firefly in August. |
Pipet tips touched with bare hands | + | ND | Positive. |
Saliva | + | ND | Positive. |
* ND = Not determined.
** 100 µl of RNase-free water was placed in the palm of the subject's bare hand. 5 µl of that water was used in the assay according to the kit protocol.
Enzymes* Tested with the RNaseAlert® Technology | Before Addition of RNase A* | After Addition of RNase A* | Comments |
---|---|---|---|
BamH1, 100 U | - | +++ | |
DNase I, 10 U | - | +++ | |
E. coli S30 extract, 1/10th Volume | + | ND** | |
Hind III, 100 U | - | +++ | |
Klenow Fragment (Exo-) (50 U) | - | +++ | |
Kpn I | - | +++ | |
Msp I | - | +++ | |
Mung Bean Nuclease | + | ND | |
Nco I | - | +++ | |
Nde I | - | +++ | |
Nuclease-free water | - | +++ | |
Proteinase K | - | +++ | |
RNase A | +++ | ND | |
RNase I | +++ | ND | |
RNase T1 | +++ | ND | |
S1 nuclease | +++ | ND | |
Sac I | - | +++ | |
Sal I | - | +++ | |
Shrimp Alkaline Phosphatase | - | +++ | |
SuperTaq | - | +++ | |
T3 RNA Polymerase / placental ribonuclease inhibitor protein mix, 100 U / 30 U | - | +++ | |
T4 Polynucleotide Kinase | - | +++ | |
T4 RNA Ligase | - | +++ | |
T7 RNA Polymerase / placental ribonuclease inhibitor protein mix, 100 U / 30 U | - | +++ | |
Xba I | - | +++ |
*Tested at 10%
** ND = Not determined.