Protocol for Co |
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Reagents required: 1. NEB HiScribe™ T7 High Yield RNA Synthesis Kit, NEB #E2040S 2. TriLink CleanCap® Reagent AG, N-7113, or CleanCap® Reagent AG (3’ OMe), N-7413 Reagents not supplied: 1. Double-stranded DNA template **NOTE: CleanCap AG requires modification to the sequence just downstream of the T7 promoter sequence, replacing the “GG” that follows the T7 promoter sequence with an “AG” as follows: Standard T7 promoter (underlined) with GG: 5’ TAATACGACTCACTATAGG 3’ Sequence required for CleanCap® with AG: 5’ TAATACGACTCACTATAAG 3’ 2. NEB Monarch® RNA Cleanup Kit (500 µg), T2050 Co-transcriptional Capping Protocol: (NOTE: 5 mM final each NTP instead of 10 mM in standard HiScribe Reaction) Thaw the necessary components, keep the T7 RNA Polymerase Mix on ice. Mix and pulse-spin in a microfuge to collect the solutions to the bottom of the tubes. Set up the reaction at room temperature in the following order:Component Volume Concentration Nuclease-free Water X µl
10X Reaction Buffer 2 µl 0.5X final 100 mM ATP 2 µl 5 mM final 100 mM GTP 2 µl 5 mM final 100 mM UTP 2 µl 5 mM final 100 mM CTP 2 µl 5 mM final 100 mM CleanCap® AG or AG (3’ OMe) (N-7113 or N-7413) 1.6 µl 4 mM final Linear Template DNA X µl 1 µg total T7 RNA Polymerase Mix 4 µl
Total 40 µl Mix thoroughly by pipet and pulse-spin in a microfuge. Incubate at 37°C for 2 hours in a dry air incubator or PCR machine to prevent evaporation. Follow the protocol provided with the HiScribe T7 High Yield RNA Synthesis Kit (NEB #E2040S) for DNase treatment and RNA purification. |
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