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Team:SDU-Denmark/labwork/Protocols/revtrans
From 2012.igem.org
| mRNA Isolation | PCR | Miniprep | Check Digest |
| 3A-Assembly | Colony-PCR | Transformation | Gel-electrophoresis |
| Reverse Transcriptase | Mutagenisis | PCR-,gel clean-up | Content |
Reverse Transcriptase PCR
Qiagen ®
1) Thaw template RNA, primer solutions, dNTP Mix, 5x QIAGEN OneStep RT-PCR Buffer, and RNase-free water, and place them on ice. It is important to mix the solutions completely before use to avoid localized differences in salt concentration. 2) Prepare a master mix according to the table. The master mix typically contains all the components required for RT-PCR except the template RNA. Prepare a volume of master mix 10% greater than that required for the total number of reactions to be performed. A negative control (without template RNA) should be included in every experiment.
Component Volume/reaction Final concentration Master mix RNase-free water (provided) Variable - 5x QIAGEN OneStep RT-PCR Buffer 10.0 μl 1x dNTP Mix (containing 10 mM of each dNTP) 2.0 μl 400μl of each dNTP Primer A Variable 0.6 μM† Primer B Variable 0.6 μM† QIAGEN OneStep RT-PCR Enzyme Mix 2.0 μl – RNase inhibitor (optional)‡ Variable 5–10 units/reaction Template RNA Template RNA, added at step 4 Variable 1 pg – 2 μg/reaction Total volume 50.0 μl –3) Mix the master mix thoroughly, and dispense appropriate volumes into PCR tubes. Mix gently, for example, by pipetting the master mix up and down a few times. 4) Add template RNA ( 2 µg/reaction) to the individual PCR tubes. The QIAGEN OneStep RT-PCR Kit can be used with total RNA, messenger RNA, or viral RNA. 5) When using a thermal cycler with a heated lid, do not use mineral oil. Proceed directly to step 6. Otherwise, overlay with approximately 50 µl mineral oil. 6)Program the thermal cycler according to the program and start the RT-PCR program while PCR tubes are still on ice. Wait until the thermal cycler has reached 50°C. Then place the PCR tubes in the thermal cycler.
