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| == Barcoded cDNA generation with fusion PCR. == | == Barcoded cDNA generation with fusion PCR. == | ||
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| - | #### Material | ||
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| #### Protocol | #### Protocol | ||
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| List of paramater tested. | List of paramater tested. | ||
| - | | RT | - | - | + | + | - | - | + | + | | + | | RT | + | + | | Memo _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ | | |
| - | ^ Tm/Target gene | MORF4L1 | MORF4L1 ^ MORF4L1 ^ MORF4L1 | MRFAP1 | MRFAP1 ^ MRFAP1 ^ MRFAP1 ^ | + | ^ Tm/Target gene ^ MORF4L1 ^ MRFAP1 | | | | |
| - | ^ Row/Col on PCR plate | 1 | 2 ^ 3 ^ 4 | 5 | 6 ^ 7 ^ 8 ^ | + | ^ Row/Col on PCR plate ^ 1 ^ 2 | | | | |
| - | ^ Frd primer | PI 11 | PI 11 | PI 11 | PI 11 | PI 13 | PI 13 | PI 13 | PI 13 | | + | ^ Frd primer | PI 11 | PI 13 | | | | |
| - | ^ Rvs Primer | PI 2 | PI 2 | PI 2 | PI 2 | PI 2 | PI 2 | PI 2 | PI 2 | | + | ^ Rvs Primer | PI 2 | PI 2 | | | | |
| - | | Fusion PCR template | | | | | | | | | | + | | | | | | | | |
| - | | Tm | | | 56 | 56 | | | 58 | 58 | | + | | Row/Template | | | | | | |
| + | | A | 7-20 | 5-20 | | | | | ||
| + | | 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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| *Mix the following reagents in one tube (ideally from top to bottom) | *Mix the following reagents in one tube (ideally from top to bottom) | ||
| ^ Check ^ Reagent ^ Volume (µl) 1 reaction ^ Volume (µl) for 25 reactions with 20µL vol ^ Final concenration |^ Memo ^ | ^ Check ^ Reagent ^ Volume (µl) 1 reaction ^ Volume (µl) for 25 reactions with 20µL vol ^ Final concenration |^ Memo ^ | ||
| - | | | ddH20 | 6.62 | 331 | - | | | | + | | | ddH20 | 6.12 | 306 | - | | | |
| | | 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. | | ||
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| ^ 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 | | ||
| - | | | | | **Aliquot 18 µL to each tube** | | | | | + | | | | | **Aliquot 17 µL to each tube** | | | | |
| | | 10µM Frd Primer | 0.5 | 1 | 0.5 | µM | | | | | 10µM Frd Primer | 0.5 | 1 | 0.5 | µM | | | ||
| | | 10µM Rvs Primer | 0.5 | 1 | 0.5 | µM | | | | | 10µM Rvs Primer | 0.5 | 1 | 0.5 | µM | | | ||
| + | | | Fusion PCR template | 0.5 | 1 | | | | | ||
| | ^ Total (µL) ^ 10 ^ 20 | | | | | | ^ Total (µL) ^ 10 ^ 20 | | | | | ||
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| *Run the following program on a thermal cycler | *Run the following program on a thermal cycler | ||
| - | ^ 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 | 10 sec | Denaturation. | |
| - | | 3 | X ˚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 | 3 min | 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. | |
| - | | 7 | 4˚C | FOREVER | Stop reaction. | | + | | 7 | 4˚C | FOREVER | Stop reaction. | |
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