RIBOPROTECT. RNase Inhibitor RT33-020, RT33-100

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1 RIBOPROTECT RT33-020, RT33-100

2 RT33-020, RT RIBOPROTECT The RIBOPROTECT is a recombinant protein isolated and purified from Escherichia coli. It inhibits ribonuclease (RNase) activity of enzymes such as RNase A, RNase B, RNase C by non-covalent binding in a 1:1 ratio. RIBOPROTECT is intended for use in alications where the presence of RNases may present a hazard to RNA quality and experiment results, e.g. in RNA isolation, cdna synthesis, RT-PCR, in vitro transcription and translation, or RNase-free monoclonal antibody preparation. RIBOPROTECT shows no activity towards RNase 1, RNase T1, RNase T2, S1 nuclease and RNase H. DNA-Gdańsk

3 Features and advantages Completely inhibits RNase A, B and C activity No polymerase or reverse transcriptase activity DNase-, RNase- and DNA-free Stable at a wide temperature, ph (5 8) and DTT concentration ranges Compatible with the TRANSCRIPTME Reverse Transcriptase (cat. no. RT32) Alications RNA isolation and purification cdna synthesis, RT-PCR, RT-QPCR in vitro transcription and translation RNA amplification

4 RT33-020, RT RIBOPROTECT Usage The optimal final concentration of the RIBOPROTECT in a reaction depends on the level of RNase contamination, the incubation time and the compounds present in the reaction mixture. It falls within a range of 2 4 U/µl. For a standard reverse transcription reaction, use 1 µl (40 U) of the RIBOPROTECT inhibitor for a final sample volume of 20 µl. For the optimal RIBOPROTECT activity, a final DTT concentration of mm is essential. Adding the RIBOPROTECT inhibitor to the reaction before the reagents, which may cause contamination, is recommended. Using RIBOPROTECT does not exclude RNase H treatment after amplification of the first strand cdna.

5 Quality control The absence of DNase and RNase activity has been confirmed using the relevant procedures. No DNA contamination has been detected. In addition, the functional quality is tested by RT and subsequent PCR and QPCR assays. Unit definition One unit is defined as the amount of enzyme required to inhibit the activity of 5 ng RNase A by 50%. Activity is measured by the inhibition of the hydrolysis of cytidine 2,3 cyclic monophosphate by RNase A.

6 RT33-020, RT Additional information mm DTT presence is essential for optimal activity of the RIBOPROTECT The storage buffer contains 8 mm DTT, however, if the ratio of the inhibitor to the final sample volume is less than 1 : 8, then the addition of DTT to a final concentration of mm DTT is recommended. Storage buffer 20 mm Tris-HCl (ph 8.0), 50 mm KCl, 0.5 mm EDTA, 8 mm DTT, 50% (v/v) glycerol DNA-Gdańsk

7 Troubleshooting Problem Possible cause Solution No RNase Inhibitor activity RIBOPROTECT shows no activity towards the RNases present in the sample DTT concentration is too low No activity owing to denaturating conditions Maintain aseptic working conditions. Use disposable gloves, changing them as frequently as required. Use RNase-free consumables. Only work in an area assigned for working with RNA and with equipment designated for that purpose. Use a different RNase inhibitor. Add the required quantity of DTT to a final concentration of mm DTT. Avoid conditions which compromise the RIBOPROTECT activity: The enzyme activity is inhibited by denaturating agents such as SDS, urea and oxidising substances Inactivation of the enzyme occurs at 65 C.

8 RIBOPROTECT Component RT U RT U RT33-S 120 U RIBOPROTECT (40 U/µl) 50 µl 5 x 50 µl 3.3 µl Storage & shiing Storage conditions Store at -20 C. The product is stable for 12 months providing it is stored and used correctly. Shiing conditions Shiing on dry or blue ice. For research use only Date of purchase Warranty 12 months from the date of purchase DNA-Gdańsk

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