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ޤµ 4-5 ‡¦´ÊŠ ‹µ„œ´Êœ‹¹ ŠÁ˜·¤ Solution III (3 M sodium acetate pH 4.8) ž¦·¤µ˜¦ 150 ŤÇ¦¨·˜¦ Ÿ¤Ä®oÁ oµ„´œ
×¥„¨´®¨°—Åž¤µ®¨µ¥Ç ‡¦´ÊŠÂ¨o ªÂnĜœÊε ȊÁž}œÁª¨µ 10 œµš¸ ¨oªœÎµÅžž{~ œ˜„˜³„°œš¸É‡ªµ¤Á¦Èª¦° 12,000
rpm Áž} œÁª¨µ 15 œµš¸ ¨³Á„ȝnªœÄ¤µŸ¤„´ Isopropanol ž¦·¤µ˜¦ 450 ŤÇ¦¨·˜¦Â¨oªœÎ µÅžž{~œ˜„˜³„°œ¡
¨µ¤·—š¸É‡ªµ¤Á¦Èª¦° 12000 rpm Áž}œÁª¨µ 15 œµš¸ ‹µ„œ´ÊœœÎµ˜³„°œš¸Éŗo¤µ¨o µŠ—oª¥ 70% ethanol ž¦·¤µ˜¦
1 ¤·¨¨·¨·˜¦Â¨³˜´ÊŠš·ÊŠÄ®o ˜³„°œÂ®oŠš¸É°»–®£¼¤·®o °ŠÁž} œÁª¨µž¦³¤µ– 15 œµš¸Â¨oª¨³¨µ¥˜³„°œ—oª¥ TE buffer (10
mM Tris-HCl ¨³ 1 mM EDTA pH 8.0) ¨o ªœÎµÅžÄ®o‡ªµ¤¦o°œš¸É 65qC Áž}œÁª¨µ 10 œµš¸ ¨³Á„ȝ¦´ „¬µÅªoš¸É
°»–®£¼¤· -20qC ®¦º°œÎµ¤µÄoŗo š´œš¸
3. „µ¦œÎµ¡¨µ¤·—¤µª·ÉŠÄœª»oœ°³„µÃ¦£µ¥Ä˜o„¦³ÂÅ¢¢jµ
3.1 „µ¦Á˜¦¸¥¤ 0.7% ª»oœ°³„µÃ¦
¨³¨µ¥ŸŠª»oœ°³„µÃ¦ 0.7 „¦´¤Äœ Tris-borate-EDTA (TBE buffer : 89 mM Tris-HCl, 89 mM Boric acid
¨³ 25 mM EDTA pH 8.0) ž¦·¤µ˜¦ 100 ¤¨. —oª¥‡ªµ¤¦o°œ ¨oª˜´ÊŠš·ÊŠÅªoÄ®o°»–®£¼¤·¨—¨ŠÁ®¨º°ž¦³¤µ– 60qC
‹µ„œ´ÊœœÎµÅžÁš¨ŠÄœ tray š¸ÉªµŠ comb Ūo ¨oªÄ®o¤¸‡ªµ¤®œµÃ—¥ž¦³¤µ– 3-5 ¤·¨¨·Á¤˜¦ (¤¤.) ˜´ÊŠš·ÊŠÅªoš¸É
°»–®£¼¤·®o°Šž¦³¤µ– 30-45 œµš¸ Á¡ºÉ°Ä®oª»oœ°³„µÃ¦Â Ȋ˜´ª ‹µ„œ´Êœ—¹Š comb °°„‹µ„ª»oœ°³„µÃ¦‹³Á„·—Áž}œn °Š
Ūo 宦´ Än µ¦¨³¨µ¥˜´ª°¥nµŠ¡¨µ¤· —
3.2 „µ¦Á˜¦¸¥¤˜´ª°¥nµŠ¡¨µ¤·—Á¡ºÉ°ª·É ŠÄœª»oœ°³„µÃ¦
Ÿ¤˜´ª°¥nµŠ¡¨µ¤·—š¸É „´—Å—o„´ loading buffer (30% glycerol, 0.05% Bromophenol blue ¨³ 5 mM
EDTA pH 8.0) ‹µ„œ´ÊœœÎµÅžÄnĜn°Š °Š 0.7% ª»o œ°³„µÃ¦ (1 n°Š˜n° 1 ˜´ª°¥nµŠ) ¨oªœÎµÅžª·É Š£µ¥Ä˜o
„¦³ÂÅ¢¢j µ 100 ꨘrĜµ¦¨³¨µ¥ TBE buffer Áž}œÁª¨µž¦³¤µ– 45 œµš¸ ‹µ„œ´ÊœœÎµÂŸnœª»oœ°³„µÃ¦Åž¥o°¤Äœ
µ¦¨³¨µ¥Á°›·Á—¸¥¤Ã¦Å¤—r‡ªµ¤Á o ¤ oœ 1 ŤÇ¦„¦´¤˜n°¤· ¨¨·¨·˜¦ Áž}œÁª¨µ 5 œµš¸ ¨oªœÎ µÅžÂnĜœÊ儨´ÉœÁž}œ
Áª¨µ 10-20 œµš¸‹µ„œ´ÊœœÎµÅž—¼Â™ °Š¡¨µ¤·—£µ¥Ä˜oÁ‡¦ºÉ°Š„εÁœ·—Š°»¨˜¦µÅªÃ°Á¨˜ (UV transilluminator:
Vilber lourmat TFX-20.M, France)
4. „µ¦œÎµ¡¨µ¤·—¨¼„Ÿ¤Á o µ¼nÁŽ¨¨rÁ‹oµoµœ
4.1 „µ¦Á˜¦¸ ¥¤ÁŽ¨¨r¡¦o°¤¦´ (competent cell)
Á ¸É¥ÁºÊ° E. coli DH5v ®¦º° INV vFc ž¦³¤µ– 8-10 Ç蜸ŞÁ¨¸Ê¥ŠÄœ°µ®µ¦Á¨¸Ê¥ŠÁºÊ° LB š¸ÉŤn¤¸¥µ
ž’·¸ªœ³ž¦·¤µ˜¦ 50 ¤¨. š¸É°»–®£¼¤· 37qC ĜÁ‡¦ºÉ°Šn¤ÁºÊ°ÂÁ ¥nµš¸É ‡ªµ¤Á¦Èª¦° 150 rpm Áž}œÁª¨µ 4 ´ÉªÃ¤Š
(¤.) ‹µ„œ´ÊœœÎµÁºÊ°ÅžÂnĜœÊε ȊÁž}œÁª¨µ 10 œµš¸ ¨oªœÎ µÅžž{~œÁ®ª¸É¥ŠÁ¡ºÉ °Á„ȝ˜³„°œš¸É‡ªµ¤Á¦Èª¦° 5000 rpm
Áž}œÁª¨µ 6 œµš¸ š¸É°»–®£¼¤· 4qC ¨oª‹¹ŠÁ˜·¤µ¦¨³¨µ¥Â‡¨ÁŽ¸¥¤˜¨°Å¦—r (10 mM Tris-HCl pH 8.0 ¨³ 50 mM
CaCl 2) š¸É nÁ¥Èœ¨ŠÅž 30 ¤¨. Á¡ºÉ°šÎµ„µ¦„¦³‹µ¥˜³„°œÁºÊ °Ã—¥Á ¥nµÁµÇ ‹µ„œ´Ê œÂnŪoĜœÊε ȊÁž}œÁª¨µ 5 œµš¸
¨oªšÎ µ„µ¦ž{~œÁ„È ˜„˜³„°œš¸É £µª³Á—·¤Â¨³Á˜·¤µ¦¨³¨µ¥Â‡¨ÁŽ¸¥¤‡¨°Å¦—r ¨ŠÅž 4 ¤¨. „¦³‹µ¥˜³„°œÁºÊ°
ÁµÇ nĜœÊε ȊÁž}œÁª¨µ 30 œµš¸ nŠÁ„ȝÁºÊ °¨ŠÄœ®¨°— microcentrifuge ®¦º°œÎµÅžÄoŗoš´ œš¸
4.2 „µ¦™nµ¥¡¨µ¤· —Á oµ¼nÁŽ¨¨r—oª¥ª·›¸ transformation
Ÿ¤¡¨µ¤·— (pBluescript II KS ®¦º° pHY300PLK ®¦º° pCHIB1 ®¦º° pHYB43) Ä®oŗo‡ªµ¤Á o ¤ oœ
ž¦³¤µ– 200 œµÃœ„¦´¤ (Ťn ‡ª¦Á„·œ 10 ¤‡.¨.) ˜n°ÁŽ¨¨r¡¦o°¤¦´ž¦· ¤µ˜¦ 200 ¤‡.¨. œÎµnªœŸ¤ÅžÂnĜœÊε Ȋ
Áž}œÁª¨µ 30 œµš¸ ‹µ„œ´ÊœœÎµÅžÂnĜ°n µŠœÊε¦o°œš¸É°»–®£¼¤· 42qC Áž}œÁª¨µ 90 ª·œµš¸ ¨³œÎµ„¨´¤µÂnĜœÊε Ȋ
š´œš¸Áž}œÁª¨µ 5 œµš¸ ‹µ„œ´ÊœÁ˜· ¤°µ®µ¦Á¨¸Ê¥ŠÁºÊ°Á®¨ª LB ¨ŠÅžž¦·¤µ˜¦ 800 ¤‡.¨. œÎµÅžn¤Á¨¸Ê¥ŠÄœ°n µŠœÊε¦o°œš¸É