iGEM 2013 — Team Nanjing-China

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# Team:Nanjing-China/Notebook

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| --- | --- | --- | --- | --- | --- | --- | --- | --- |

|  |
| --- |
| Contents   * [1 1. Extraction of mRNA](#1._Extraction_of_mRNA) * [2 2. RT-PCR](#2._RT-PCR) * [3 3.PCR](#3.PCR)   + [3.1 (1) Colony PCR](#.281.29_Colony_PCR)   + [3.2 (2) PCR with DNA as template](#.282.29_PCR_with_DNA_as_template) |

### 1. Extraction of mRNA

:   Kit

### 2. RT-PCR

:   Kit

### 3.PCR

#### (1) Colony PCR

:   |  |  |
    | --- | --- |
    | 2×PrimeStar | 12.5μl |
    | Forward primer | 1~2.5μl |
    | Reverse primer | 1~2.5μl |
    | Bacteria solution | 1~2μl |
    | dd water | up to 25μl |

:   |  |  |
    | --- | --- |
    | Step 1 | 94℃ |
    | Step 2 | 94℃ |
    | Step 3 | Annealing temperature |
    | Step 4 | 72℃ |
    | Step 5 | goto step2, 25~35 cycles |
    | Step 6 | 72℃ |
    | Pause at 4℃ |  |

#### (2) PCR with DNA as template

:   |  |  |
    | --- | --- |
    | 2×PrimeStar | 12.5μl |
    | Forward primer | 1μl |
    | Reverse primer | 1μl |
    | Template DNA | 0.5μl |
    | dd water | up to 25μl |

:   |  |  |
    | --- | --- |
    | Step 1 | 94℃ |
    | Step 2 | 94℃ |
    | Step 3 | Annealing temperature |
    | Step 4 | 72℃ |
    | Step 5 | goto step2, 25~35 cycles |
    | Step 6 | 72℃ |
    | Pause at 4℃ |  |

:   Gel running and purify the product.

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