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In vitro Transcription

0.5 M EDTA, pH 8.0

Product information

Applications

• Termination of metal dependent enzymatic reactions.
• Protection of DNA against metal dependent nucleases.

Description

An aqueous solution of 0.22 µm membrane-filtered ethylendiaminetetraacetic acid (EDTA), sodium salt. Ideal for biochemistry or molecular biology applications that require a chelator for divalent cations. By forming complexes with metal ions, EDTA inhibits metal-dependent enzymatic reactions. EDTA complexes metal ions at a 1:1 molar ratio.

Nucleic acids are normally stored in TE buffer that contains 1 mM EDTA, which prevents degradation of nucleic acids by metal dependent nucleases.

Quality Control

The absence of endo-, exodeoxyribonucleases and ribonucleases confirmed by appropriate quality tests.

Storage

Store at 4°C.

Patents, Licenses, Trademarks

Protocols & recommendations

RECOMMENDATIONS FOR USE

ADDITIONAL PROTOCOLS

Purification of RNA Transcripts

• Template DNA: may interfere with downstream applications of the RNA transcript. Template DNA should be removed by DNase I digestion directly after the transcription reaction. Add 2 u of DNase I, RNase-free, mix and incubate at 37°C for 15 min, then add 2 µl of 0.5 M EDTA, pH 8.0 and incubate at 65°C for 10 min to stop the reaction.
• Proteins and nucleotides: phenol/chloroform extraction and ethanol precipitation of RNA transcripts is recommended.

1. To 50 µl reaction mixture, add 85 µl of DEPC-treated water and 15 µl of 3 M Sodium Acetate. Mix thoroughly.
2. Extract with an equal volume of 1:1 phenol/chloroform mixture, and then twice with an equal volume of chloroform. Collect the aqueous phase and transfer to a new tube.
3. Precipitate the RNA by adding 2 volumes of ethanol. Incubate at -20°C for at least 30 min and collect the pellet by centrifugation.
4. Remove the supernatant and rinse the pellet with 500 µl of cold 70% ethanol.
5. Resuspend the RNA in 20 µl of DEPC-treated water.
6. Store the RNA at -20°C or -70°C.