ࣛ࢓ࣜࢰ࢕࣑ PCR Mx3000P ࡡ౐⏕Ἰណ஥㡧➴
㸟 ࢪ࣭࢜ࢹࡡ࡝࠷ࢲ࣭ࣖࣇ㸝㸭㏻ࢲ࣭ࣖࣇ➴㸞ࢅࢬࢴࢹࡌࡾ᫤ࡢࠉ࿔ࡽ࠾ࡼࡡකࡡ⁻ࡿ㎲ࡲ
ࢅ㐵᩷ࡌࡾࡒࡴ࡞௛ᒌࡡ㯦࠷ᯗ*ࢅᮇమ࡞ࡢࡴ࡙ࡂࡓࡈ࠷ࠊ㯦࠷ᯗ࠿࡝࠷ሔྙࡢ࡚ࡀࡾࡓࡄ୯
ኳ࡞ᐞࡎ࡙ῼᏽࡊ࡙ࡂࡓࡈ࠷㸝አഁ⛤⁻ࡿࡒක࡝࡜࡞ࡻࡽㄏᕣ࠿⏍ࡋࡷࡌࡂ࡝ࡾࡒࡴ㸞ࠊ
㸟㯦࠷ᯗ࠿⾔᪁୘᪺࡚ࡌࠊᚨᙔࡒࡽࡡ࠵ࡾ᪁ࡢ㏻⤙ࢅࡂࡓࡈ࠷ࠊ
㸟ࣚࣤࣈࡢᾐ⪎ဗ࡚ࡌࠊ↋㥇࡞ᾐ㈕ࡊ࡝࠷ࡻ࠹࡞ Run ⤂஡ᚃࡢࣚࣤࣈ࠿วࡿࡾࡻ࠹࡞スᏽࡊ
࡙ࡂࡓࡈ࠷ࠊ
スᏽ᪁Ἢ㸞Run ࢅࢪࢰ࣭ࢹࡌࡾ㝷࡞ୖオࢅ᧧షࡊࡱࡌ
Thermal Profile Setup ࢪࢠ࣭ࣛࣤЍStart Run ࣍ࢰ
ࣤЍ㛜࠷ࡒ Run Status ࢱ࢕࢓ࣞࢡ࣍ࢴࢠࢪࡡୌ␊ୖ࡞
࠵ࡾࠖTurn lamp off at end of runࠗ࡞ࢲ࢘ࢴࢠࢅථࡿ
ࡾࠊࢲ࢘ࢴࢠࢅථࡿࡒࡼࠖStartࠗࢅᢪࡊ࡙ OK.
㸟౐⏕᫤࡞୒࠿ୌ PC ࠿ࣆ࣭ࣛࢫࡊ࡙ࡵᮇమࡡ Run ࡞ࡢᙫ㡢ࡢ࠵ࡽࡱࡎࢆࠊࡱࡒࠉRun ࡡࢸ
࣭ࢰ࣭㸝1run ฦࡡࡲ㸞ࡢᮇమ࡞オ᠀ࡈࡿ࡙࠷ࡱࡌࡡ࡚ࠉPC ࢅᚗ᪟ࡊࡒᚃࠉᮇమࡡࢸ࣭ࢰ࣭
ࢅ࿣ࡦฝࡊ࡙ᨾฝ࡚ࡀࡱࡌࠊ
ࢸ࣭ࢰ࣭ᨾฝ᪁Ἢ㸞PC ࢅ්㉫ິࡊࡒᚃࠉInsturumentЍୌ␊ୖࡡ Download Data....
ࢅ㐽ᢝࠊ࡝࠽ࠉᮇమࡡࢸ࣭ࢰ࣭ࡢ㸦ࡗࡊ࠾ṟࡎ࡝࠷ࡡ࡚ࠉᨾฝࡊࡒ࠷ࢸ࣭ࢰ࣭ࡢḗࡡ
Run ࡚୕᭡ࡀࡈࡿࡾ๑࡞ࡌࡃ࡞ᨾฝࡊ࡙ࡂࡓࡈ࠷ࠊ
㸟Finish ࡌࡾ㝷ࠉࢦࣤࣈࣜࢅཱིࡽฝࡊࠉࣚࣤࣈ࠿Ⅴ࠷࡙࠷࡝࠷஥ࢅ☔ヾࡊ࡙ࡂࡓࡈ࠷ࠊࣚࣤ
ࣈ࠿Ⅴ࠷࡙࠷ࡾሔྙࡢࠉPC ࡡࣚࣤࣈ࣍ࢰࣤࢅࢠࣛࢴࢠࡊวࡖ࡙࠽ࡂࡆ࡛ࠊ
㸱モⷾ➴ࡡ᝗ሒ㸳
㸟ࢰ࢜ࣚࡡ kit ࡢࠉ▯࠷㒼า࡚ࡵࡒࡂࡈࢆࡡ Dye ࠿ථࡾࡡ࡚᳠ฝវᗐ࠿ࡻ࠷࠿㛏࠷㒼าࡢቌ
ࡷࡊ࡞ࡂ࠷ࠊୌ᪁ࠉࢪࢹࣚࢰࢩ࣭ࣤࡷ ABI ࡢࠉវᗐࡢᘽࡴ㸴ࡓ࠿ 1 kbp ࡚ࡵቌࡷࡎࡾࡼࡊ࠷
㸟㸩Υ࡞⁈よࡊࡒ master mix ࡡಕᏋ᭿㝀ࡢ⣑ 1 ࣧ᭮ࠊᑚฦࡄࡊ්࡙෼⤎ࡊ࡙࠽ࡂ࡛ࡻ࠷ࠊ
㸟ࢲ࣭ࣖࣇ㢦ࡢ ABI ࡞ᑊᚺࡊࡒࡵࡡ࡚࠵ࡿࡣ౐࠻ࡱࡌ㸝ABI ࡛ྜྷࡋぞ᰹࡞࡝ࡖ࡙࠷ࡾࡒࡴ㸞
㸟well ࡞ථࡿࡾ Minimum volume ࡢ 10 ȣl㸝1 well ࡡῼᏽ࡞᭩఩㸨࢜ᡜࡢᚪさ࡚ࠉࡆࡿࢅ
‫ ࡌࡒ‮‬well ࡡ⾪㟻✒࠿ 10 ȣl ࡚㝀⏲࡞࡝ࡾࡒࡴ㸞࡚࠵ࡾ࠿ࠉౚ࠻ࡣ 5ȣl ࡞ࡊ࡙࣐ࢾ࢛ࣚࣜ
࢕ࣜࢅ 10ȣl ㊂ࡌ⿤ᢇࡵྊ⬗㸝ῼᏽ㟻✒ࢅ✄ࡅࡾࡒࡴ㸞ࠊࡊ࠾ࡊࠉࢦࣤࣈࣜ volume ࢅୖࡅࡾ
࡛ㄏᕣ࠿ኬࡀࡂ࡝ࡾࡡ࡚Ἰណࡢᚪさ㸝᮪௲᳠ゞࡡ㝷࡞ฺ⏕ࡌࡾࡡࡢ OK ࠾ࡵࡊࡿ࡝࠷㸞ࠊ
௧ୖ 16ȣl ࡚ࡡౚࢅ⣺௒ࡌࡾࠊ
FullVelocity SYBR Green QPCR Master Mix ࢅ౐⏕ࡊࡒ Volume 16 ȣl ࡚ࡡౚ
Template (1, 1/2, 1/4, 1/8.....)
1 ȣl
㸝10 ng total RNA /ȣl ⏜ᮮࡡ cDNA ࣚ࢕ࣇ࣭ࣚࣛ㸞
10 ȣM Primer ࢬࢴࢹ
2x master mix
0.16ȣl ࡍࡗ
8 ȣl
1/500 Reference Dye
0.24 ȣl
ኬሪⵠ⏻Ề
6.44 ȣl
㸱ࢦࣤࣈࣜష⿿᫤ࡡἸណ஥㡧㸳
㸟ᖲᆍೋࢅฝࡌሔྙࠉㄏᕣࢅ᭩ᑚ㝀࡞ᢒ࠻ࡾࡒࡴࠉྜྷࡋ᮪௲ࡡࢦࣤࣈࣜࡢ template, primer,
master mix, Dye ➴ࡌ࡬࡙ࡢࡋࡴ࡞Ίࡏࡒࡵࡡࢅష⿿ࡊࠉࡐࡿࢅฦࡄ࡙ථࡿࡾࡻ࠹࡞ࡌࡾࠊ
㸟ࢱ࢕࣏࣭࠿⏍ࡋࡒሔྙ➴ࡢࠉ࢓ࢼ࣭ࣛࣤࢡῺᗐࡡ᮪௲ࢅ࠷ࢀ࠷ࢀ᳠ゞࡌࡾࡻࡽࡵࣈࣚ࢕࣏
࣭ࢅషࡽ├ࡊࡒ᪁࠿⤊ῥⓏ࡛ᛦࢂࡿࡾࠊ