Team:SDU-Denmark/labwork/Protocols/revtrans
From 2012.igem.org
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RNA) should be included in every experiment.</br> | RNA) should be included in every experiment.</br> | ||
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Revision as of 14:56, 16 September 2012
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
- 5 ul Fastdigest greenbuffer / NEB Buffer 2
- 0.5 ul BSA
- 0.5 ul EcoRI-H
- 0.5 ul PstI
- 19 ul dH20 (0,5 ul less when using BSA)