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Use of "bad" nucleotides can result in smeared products and poor incorporation
Radiolabeled nucleotides are commonly used in in vitro transcription, random priming, and primer extension reactions to generate labeled RNA and DNA probes. During kit development and testing in our research labs, Ambion samples many lots of numerous radiolabeled nucleotides. Through this research, we have noticed that radiolabeled nucleotides vary in their stability from lot to lot and supplier to supplier. Use of "bad" nucleotides can result in smeared products and poor incorporation. These symptoms may also make a researcher suspicious of the labeling kit or reagents he or she is using. Therefore, we suggest setting up a "cold" reaction (unlabeled) to distinguish between a bad kit and degraded nucleotides producing inhibitors. If the cold reaction produces products such as those shown in Figures 1 and 2, then there is either not enough of the labeled "limiting" nucleotide in the reaction or it has gone bad. Such evidence of a bad nucleotide is usually enough to request a replacement of the labeled nucleotide from the supplier.
"Cold Reaction" Procedure
Random priming
When using Ambion's DECAprime IIí Random Priming Kit (Catalog #1455), a "cold" reaction is set up by mixing equal amounts of the dNTP-dATP Mix (2.5 µl) and the dNTP-dCTP Mix (2.5 µl) in the 25 µl reaction volume. Under these conditions there will be at least 1 nmole of dNTPs present (e.g. 10 µM final concentration of each dNTP in a 25µl reaction or 250 nmoles each), and 200-300 ng of product should be synthesized.
The entire reaction is run in a single lane on a 3% agarose gel and is detected by EtBr staining. Random priming reactions will generate a diffused band or smear between 300-500 nt long. This is because product size will vary depending on where the random primers hybridize to the template sequence. The template is usually not visible since only about 25-50 ng are added to the reaction. Figure 1 shows the appearance of "unlabeled" random priming reaction products when separated on a 3% agarose gel.
In vitro transcription
When using Ambion's MAXIscriptí in vitro Transcription Kit (Catalog #1308-1326), an unlabeled or "cold" reaction is set up by adding 1 µl each of the four rNTPs in a final reaction volume of 20 µl. Under these conditions there will be 40 nmoles of rNTPs present (e.g. 500 µM final concentration of each rNTP in a 20 µl reaction, or 10 nmoles each), and 6-10 µg of product should be synthesized. In vitro transcription reactions will generate a single band on a denaturing agarose or acrylamide gel. On a nondenaturing agarose gel, the transcription product is also usually a single band although secondary structure can sometimes cause it to be diffuse or resolve into multiple products. The DNA template can be left in the reaction or destroyed by treatment with DNase immediately after transcription. Products are visualized by EtBr staining. Figure 2 shows the appearance of an in vitro transcription product and template when separated on a 2% nondenaturing agarose gel. Note that the same mass of RNA will not stain as brightly with EtBr as DNA.
When using Ambion's DECAprime IIí Random Priming Kit (Catalog #1455), a "cold" reaction is set up by mixing equal amounts of the dNTP-dATP Mix (2.5 µl) and the dNTP-dCTP Mix (2.5 µl) in the 25 µl reaction volume. Under these conditions there will be at least 1 nmole of dNTPs present (e.g. 10 µM final concentration of each dNTP in a 25µl reaction or 250 nmoles each), and 200-300 ng of product should be synthesized.
The entire reaction is run in a single lane on a 3% agarose gel and is detected by EtBr staining. Random priming reactions will generate a diffused band or smear between 300-500 nt long. This is because product size will vary depending on where the random primers hybridize to the template sequence. The template is usually not visible since only about 25-50 ng are added to the reaction. Figure 1 shows the appearance of "unlabeled" random priming reaction products when separated on a 3% agarose gel.
Figure 1. Unlabeled Random Priming Reaction as Troubleshooting Procedure. Random-primed DNA was synthesized in the absence of labeled dNTP following Ambion's DECAprime™ II Kit protocol. The reaction contained 25 ng of pT7-18S rRNA linearized plasmid template (the kit control) and 2.5 µl each of the -dATP and -dCTP 5X Reaction Buffers. The reaction products were separated on a 3% agarose gel containing EtBr.
In vitro transcription
When using Ambion's MAXIscriptí in vitro Transcription Kit (Catalog #1308-1326), an unlabeled or "cold" reaction is set up by adding 1 µl each of the four rNTPs in a final reaction volume of 20 µl. Under these conditions there will be 40 nmoles of rNTPs present (e.g. 500 µM final concentration of each rNTP in a 20 µl reaction, or 10 nmoles each), and 6-10 µg of product should be synthesized. In vitro transcription reactions will generate a single band on a denaturing agarose or acrylamide gel. On a nondenaturing agarose gel, the transcription product is also usually a single band although secondary structure can sometimes cause it to be diffuse or resolve into multiple products. The DNA template can be left in the reaction or destroyed by treatment with DNase immediately after transcription. Products are visualized by EtBr staining. Figure 2 shows the appearance of an in vitro transcription product and template when separated on a 2% nondenaturing agarose gel. Note that the same mass of RNA will not stain as brightly with EtBr as DNA.
Figure 1. Unlabeled Random Priming Reaction as Troubleshooting Procedure. Random-primed DNA was synthesized in the absence of labeled dNTP following Ambion's DECAprime™ II Kit protocol. The reaction contained 25 ng of pT7-18S rRNA linearized plasmid template (the kit control) and 2.5 µl each of the -dATP and -dCTP 5X Reaction Buffers. The reaction products were separated on a 3% agarose gel containing EtBr.