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interactome:experiments:20191220 [2019/12/20 09:35] dan created |
interactome:experiments:20191220 [2019/12/20 09:45] (current) dan |
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| List of paramater tested. | List of paramater tested. | ||
| - | | RT | + | + | | Memo _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ | | | + | *10 different barcodes (purified plasmid sample) x 2 barcode types |
| - | ^ Tm/Target gene ^ MORF4L1 ^ MRFAP1 | | | | | + | *MORF4L1-40bp overlap for all genes |
| - | ^ Row/Col on PCR plate ^ 1 ^ 2 | | | | | + | *5 pilot genes with 2 barcodes each. |
| - | ^ Frd primer | PI 11 | PI 13 | | | | | + | |
| - | ^ Rvs Primer | PI 2 | PI 2 | | | | | + | |
| - | | | | | | | | | + | |
| - | | Row/Template | | | | | | | + | *Frd primer : BCamplif-Frd-MORF4L1-v2-4;PI-34-BClib-reamp-MRFAP1-Frd;PI-35-BClib-reamp-BCL2L2-Frd;PI-36-BClib-reamp-BIK-Frd;PI-37-BClib-reamp-MRFAP1L1-Frd |
| - | | A | 7-20 | 5-20 | | | | | + | *Rvs primer : PI-33-Barcode-reamp1-Rvs |
| - | | B | 7-30 | 5-30 | | | | | + | |
| - | | C | 7-40 | 5-40 | | | | | + | |
| - | | D | 7-50 | 5-50 | | | | | + | |
| - | | E | 8-20 | 6-20 | | | | | + | |
| - | | F | 8-30 | 6-30 | | | | | + | |
| - | | G | 8-40 | 6-40 | | | | | + | |
| - | | H | 8-50 | 6-50 | | | | | + | |
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| | | Phusion HF buffer (5x) | 2.0 | 100 | - | | | | | | Phusion HF buffer (5x) | 2.0 | 100 | - | | | | ||
| | | 25mM dNTPs | 0.08 | 4 | 0.2 | mM | Equal volume mix of 100mM dA/T/G/CTPs will give this solution. | | | | 25mM dNTPs | 0.08 | 4 | 0.2 | mM | Equal volume mix of 100mM dA/T/G/CTPs will give this solution. | | ||
| - | | | cDNA template | 1 | 50 | | | Diluted cDNA sample. | | + | | | DNA tempalte (10ng/µL) | 1 | 50 | | | Diluted plasmid DNA sample. | |
| ^ Transfer the tube on ice ||||||| | ^ Transfer the tube on ice ||||||| | ||
| | | Phusion pol | 0.2 | 10 | - | | Home brew Phusion pol | | | | Phusion pol | 0.2 | 10 | - | | Home brew Phusion pol | | ||
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| | | Fusion PCR template | 0.5 | 1 | | | | | | | Fusion PCR template | 0.5 | 1 | | | | | ||
| | ^ Total (µL) ^ 10 ^ 20 | | | | | | ^ Total (µL) ^ 10 ^ 20 | | | | | ||
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| ^ STEP ^ Action ^ Time ^ Memo ^ | ^ STEP ^ Action ^ Time ^ Memo ^ | ||
| | 1 | 98˚C | 3 min | Initial denaturation. | | | 1 | 98˚C | 3 min | Initial denaturation. | | ||
| - | | 2 | 98˚C | 10 sec | Denaturation. | | + | | 2 | 98˚C | 30 sec | Denaturation. | |
| | 3 | 65 ˚C | 30 sec | Annealing. See Tm table | | | 3 | 65 ˚C | 30 sec | Annealing. See Tm table | | ||
| - | | 4 | 72˚C | 3 min | Extension. **15sec/kbp, but using longer** | | + | | 4 | 72˚C | 30 sec | Extension. **15sec/kbp, but using longer** | |
| | 5 | GOTO 2 | x 29 | | | | 5 | GOTO 2 | x 29 | | | ||
| | 6 | 72˚C | 5:00 | Final extension. | | | 6 | 72˚C | 5:00 | Final extension. | | ||