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| - | == Re-trying with additional cycle to amplify MORF4L1. The annealing temp is set to 52C. == | + | == Re-trying with new cDNA sample. == |
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| - | ##Overview | + | |
| - | ^ Step ^ Description ^ Date/Initials ^ Link protocol ^ Link results ^ | + | |
| - | | 1 | Total RNA extraction from HEK293T cells | 20191122DEY | | | | + | |
| - | | 2 | 1st strand cDNA synthesis | 20191122DEY | | | | + | |
| - | | **3** | **Target gene amplification from cDNA sample** | Current | | | | + | |
| - | | 4 | BP-reaction | | | | | + | |
| - | | 5 | Sanger sequence check | | | | | + | |
| #### Material | #### Material | ||
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| - | ^ Check ^ Reagent ^ Supplier ^ Cat. no | Primer/Sample ID ^ Memo ^ | ||
| - | | | Nuclease free ddH2O | Invitrogen | 10977-015 | | | | ||
| - | | | Phusion HF buffer (5x) | NEB | B0518S | | | | ||
| - | | | 25mM dNTPs | TaKaRa | | | | | ||
| - | | | Frd Primer | FASMAC | | | PI11 to PI20 | | ||
| - | | | Rvs Primer | FASMAC | | | PI11 to PI20 | | ||
| - | | | Phusion pol | - | KM2 | | Home brew Phusion pol | | ||
| - | | | Template cDNA | | - | | Prepared on 20191122. Sample1 | | ||
| - | | | Agarose | RIKAKEN | RSV-AGRP-500G | | | | ||
| - | | | 1x TBE buffer | - | - | | Homebrew (link:) | | ||
| - | | | ExcelBand 100 bp DNA Ladder | SMOBiO | DM2100 | | | | ||
| - | | | 5x Loading dye | - | - | | Homebrew (link:) | | ||
| - | | | Midori green Advance | Nippon Genetics | NE-MG04 | | | | ||
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| - | Before preparing: Check your primer Tm at NEB website (http://tmcalculator.neb.com/#!/main with Phusion/HF buffer setting) | ||
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| #### Protocol | #### Protocol | ||
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| List of paramater tested. | List of paramater tested. | ||
| - | | RT | | + | - | + | - | + | - | + | - | + | - | | + | | RT | - | + | - | + | - | + | - | + | - | + | |
| - | ^ Tm/Target gene ^ ^ MORF4L1 ^ MORF4L1 ^ MRFAP1 ^ MRFAP1 ^ BCL2L2 ^ BCL2L2 ^ BIK ^ BIK ^ MRFAP1L1 ^ MRFAP1L1 ^ | + | ^ Tm/Target gene ^ MORF4L1 ^ MORF4L1 ^ MRFAP1 ^ MRFAP1 ^ BCL2L2 ^ BCL2L2 ^ BIK ^ BIK ^ MRFAP1L1 ^ MRFAP1L1 ^ |
| - | ^ Row/Col on PCR plate |^ 1 ^ 2 ^ 3 ^ 4 ^ 5 ^ 6 ^ 7 ^ 8 ^ 9 ^ 10 ^ | + | ^ Row/Col on PCR plate ^ 1 ^ 2 ^ 3 ^ 4 ^ 5 ^ 6 ^ 7 ^ 8 ^ 9 ^ 10 ^ |
| - | ^ 52.0 ^ A ^ | | | | | | | | | | | + | ^ Frd primer | PI 11 | PI 11 | PI 13 | PI 13 | PI 15 | PI 15 | PI 17 | PI 17 | PI 19 | PI 19 | |
| - | + | ^ Rvs Primer | PI 12 | PI 12 | PI 14 | PI 14 | PI 16 | PI 16 | PI 18 | PI 18 | PI 20 | PI 20 | | |
| - | + | | Tm | 56 | 56 | 58 | 58 | 65 | 65 | 56 | 56 | 57 | 57 | | |
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| *Mix the following reagents in one tube (ideally from top to bottom) | *Mix the following reagents in one tube (ideally from top to bottom) | ||
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| - | --- //[[daney@sfc.keio.ac.jp|Dan Yamamoto-Evans]] 2019/11/25 14:48// | + | --- //[[daney@sfc.keio.ac.jp|Dan Yamamoto-Evans]] 2019/12/10 11:37// |
| **Print at 96%** | **Print at 96%** | ||