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充分溶解并混勻PCR反應(yīng)所需的各種溶液。將2x 長(zhǎng)片段PCR 擴(kuò)增 預(yù)混液( long fragment PCR Master Mix)置于冰浴上或冰盒內(nèi)。參考下表在冰上設(shè)置PCR反應(yīng):
Component | 20uL RXN | 25uL RXN | 50uL RXN | Final Concentration |
2x 長(zhǎng)片段PCR 擴(kuò)增 預(yù)混液 | 10uL | 12.5uL | 25uL | 1 × |
10uM Forward Primer | 0.4uL | 0.5uL | 1.0uL | 0.2uM |
10uM Reverse Primer | 0.4uL | 0.5uL | 1.0uL | 0.2uM |
Template DNA | variable | variable | variable | variable |
Nuclease-Free Water | to 20uL | to 25uL | to 50uL |
Template DNA | 20uLRXN | 25uL RXN | 50uL RXN |
Genomic DNA | 4ng-0.4ug | 5ng-0.5ug | 10ng-1ug |
Plasmid or Vrial | 4pg-4ng | 5pg-5ng | 10pg-10ng |
cDNA | 4pg-0.4ug | 5pg-0.5ug | 10pg-1ug |
STEP | TEMP | TIME |
Initial Denaturation | 98°C | 30 sec |
25–35 Cycles | 98°C | 10 sec |
50–72 °C | 15 sec | |
72°C | 15-30 sec/kb | |
Final Extension | 72°C | 5 min |
Hold | 4–10 °C | ∞ |
貨號(hào) | 品名 | 規(guī)格 | 品牌 |
78DC10152-1ml | 2x 長(zhǎng)片段PCR 擴(kuò)增 預(yù)混液(long fragment PCR Master Mix) | 1ml | BIOHUB |
78DC10152-5ml | 2x 長(zhǎng)片段PCR 擴(kuò)增 預(yù)混液(long fragment PCR Master Mix) | 5ml | BIOHUB |
78DC10152-3*5ml | 2x 長(zhǎng)片段PCR 擴(kuò)增 預(yù)混液(long fragment PCR Master Mix) | 3*5ml | BIOHUB |
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