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°»–®£¼¤· 37qC Áž}œÁª¨µ 1 ¤. nŠÁºÊ°¤µž¦· ¤µ˜¦ 100 ¤‡.¨. „¦³‹µ¥¨Šœ°µ®µ¦Á¨¸Ê¥ŠÁºÊ°Â È Š LB š¸É¤¸¥µž’·¸ªœ³
°¤¡·Ž·¨¨· œ‡ªµ¤Á o¤ oœ 100 ¤‡.„./¤¨. œÎµ‹µœÁ¡µ³ÁºÊ°Åžn¤Á¡µ³š¸É°»–®£¼¤· 37qC Áž}œÁª¨µ 18-20 ¤.
5. „µ¦«¹„¬µ„µ¦‡Š°¥¼n °Š¡¨µ¤·—
5.1 „µ¦Á¨¸Ê¥ŠÁºÊ°Äœ£µª³š¸ÉŤn¤¸˜´ª„——´œ (¥µž’·¸ªœ³) ( non selective pressure)
»n¤Á¨º°„ÁºÊ°š¸É¤¸¡¨µ¤·—˜n¨³œ·—¤µœ·—¨³ 3 Ç蜸 ¤µÁ¡µ³Á¨¸Ê¥Š¨ŠÄœ°µ®µ¦Á¨¸ Ê¥ŠÁºÊ°Á®¨ª LB
ž¦·¤µ˜¦ 5 ¤¨. š¸É ¤¸¥µž’·¸ªœ³Â°¤¡·Ž·¨¨·œ‡ªµ¤Á o¤ oœ 100 ¤‡.„./¤¨. n¤Á¡µ³š¸É°»–®£¼¤· 37qC Áž}œÁª¨µ 24 ¤.
‹µ„œ´ÊœšÎµ„µ¦™nµ¥ÁºÊ° 1% ¨ŠÄœ°µ®µ¦Á¨¸Ê¥ŠÁºÊ°Á®¨ª LB ž¦· ¤µ˜¦ 5 ¤¨. š¸ÉŤn¤¸¥µž’·¸ªœ³Â°¤¡·Ž·¨¨·œÂ¨o ªšÎµ„µ¦
n¤Á¨¸Ê¥Šš¸É £µª³Á—·¤ ¨³Ä®ošÎ µÂœ¸Ê˜n°Åž‹œ‡¦ 30 ª´œ
5.2 „µ¦˜¦ª‹®µ„µ¦‡Š°¥¼n°Š¡¨µ¤·— ( segregrational stability)
nŠÁºÊ°‹µ„š»„®¨°—š¸ÉÁ¨¸Ê¥ŠÄœ£µª³ non selective pressure Ĝ˜n ¨³ª´œ¤µž¦· ¤µ˜¦ 100 ¤‡.¨. ˜n°ÁºÊ°
¨oªšÎµ„µ¦„¦³‹µ¥ÁºÊ °¨Šœ°µ®µ¦Á¨¸Ê¥ŠÁºÊ °Â Ȋ LB š¸É¤¸Â¨³Å¤n¤¸¥µž’·¸ªœ³Â°¤¡·Ž·¨¨·œÃ—¥Áš‡œ·‡ spread plate
n¤Á¡µ³š¸É°»–®£¼¤· 37qC Áž}œÁª¨µ 24 ¤. ¨oª‡Î µœª–®µ´—nªœ °ŠÃ‡Ã¨œ¸š¸É—ºÊ°¥µÂ°¤¡·Ž·¨¨·œ‹µ„¼˜¦
% ampicillin resistant colony = ‹ÎµœªœÃ‡Ã¨œ¸š¸É¹Êœœ‹µœÁ¡µ³ÁºÊ °Â Ȋ LB š¸É¤¸¥µ x 100
‹ÎµœªœÃ‡Ã¨œ¸š¸É¹Êœœ‹µœÁ¡µ³ÁºÊ°Â È Š LB š¸ÉŤn¤¸¥µ
Ÿ¨„µ¦ª·‹´¥Â¨³°£·ž¦µ¥Ÿ¨
1. „µ¦¥º œ¥´œ¡¨µ¤·—š¸ÉœÎµ¤µ«¹„¬µ
¡¨µ¤·—š¸É „´—Å—o„¨nµª‡º° pBluescript II KS ®¦º° pHY300PLK ®¦º ° pCHIB1 ®¦º° pHYB43 ¤¸œµ— 2.96,
4.87, 5.54 ¨³ 7.45 „·Ã¨Á ˜µ¤¨Îµ—´ Á¤ºÉ °˜´——oª¥Á°œÅŽ¤r˜´—‹ÎµÁ¡µ³š¸ÉÁ®¤µ³¤ (Thepouyporn, 1999;
Lertcanawanichakul, 2000) ¨³Á¤ºÉ°˜´——oª¥Á°œÅŽ¤r HindIII ‹³µ¤µ¦™Â¥„·Ê œ¥¸œÅ‡˜·Áœ°°„¤µÅ—o‹µ„¡¨µ¤·—
¨¼„Ÿ¤¥¸œÅ‡˜· ÁœpCHIB1 ¨³ pHYB43
2. Ÿ¨„µ¦«¹„¬µ segregrational stability °Š¡¨µ¤·—
¡¨µ¤·—š»„œ·—š¸ÉœÎµ¤µ«¹„¬µ¤¸ structural stability Á¤ºÉ°œÎµÁ oµ¼nÁºÊ° E. coli —´Šœ´Êœ‹¹Š«¹„¬µÁž¦¸¥Áš¸¥ÁŒ¡µ³
segregrational stability °Š¡¨µ¤·—œ·—˜n µŠÇ £µ¥ÄœÁºÊ° E. coli 2 µ¥¡´œ›»r ‡º° DH5v ®¦º° INV vFc Ž¹ÉŠÄœ
ÁºÊ°Š˜oœ¡ªn µ¡¨µ¤·— pBluescript II KS, pHY300PLK ¨³ pHYB43 ¤¸ segregrational stability Ą¨oÁ‡¸¥Š„´œ
ž¦³¤µ– 30 ª´œ ˜n¡¨µ¤·— pCHIB1 ¤¸ segregrational stability ˜Éεš¸É »—Á¤ºÉ°°¥¼nĜ E. coli š´ÊŠ 2 µ¥¡´œ›»r‡º°
ž¦³¤µ– 24 ª´œ ¨³µ¤µ¦™Â—ŠÁž°¦rÁŽœ˜r°ŠÃ‡Ã¨œ¸š¸É—ºÊ°¥µÂ°¤¡·Ž· ¨¨·œÅ—o—´Š¦¼žš¸É 1