เอกสารการประชุมวิชาการและเสนอผลงานวิจัย มหาวิทยาลัยทักษิณ ครั้งที่ 19 2552 - page 314

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Á˜¦¸¥¤ total RNA ¨³Á¡·É¤ž¦·¤µ–·Êœnªœ cDNA °Š¥¸œ REF
Á˜¦¸¥¤ total RNA ‹µ„œÊε¥µŠ °Š¥µŠ¡µ¦µµ¥¡´ œ›»r RRIM600 ˜µ¤ª· ›¸„µ¦ °Š Áž¨ºÊ°Š (2549) œÎµ total
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ATTATGGCTGAAGACGAAGACAACC-3'  ¨ ³ 5'-GGCCAATAATTCAATTGGCCCTTTATTC-3') „´  Taq
DNA polymerase (Promega, Madison, WI) š¸É°»–®£¼¤· 94 qC 2 œµš¸ 1 ¦° ˜µ¤—oª¥ 94 qC 30 ª·œµš¸, 56 qC 30
ª·œµš¸, 72 qC 30 ª·œµš¸ ‹Î µœªœ 25 ¦° ¨³ 72 qC 15 œµš¸ ‹µ„œ´Ê œœÎµ·Êœnªœ¥¸œ REF š¸É ´ŠÁ‡¦µ³®rŗo ÁºÉ°¤„´
pGEM-T Easy vector ¨³Á‡¨ºÉ °œ¥oµ¥Á oµÅžÄœÁºÊ ° E. coli JM109 ‡´ —Á¨º°„ positive clone ŞÁ˜¦¸ ¥¤¡¨µ¤·—¨³
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čo¡¨µ¤· —š¸É¤¸ insert °Š¥¸œ REF Áž}œ˜oœÂÎµ®¦´´ŠÁ‡¦µ³®r nªœ °Š open reading frame š¸É
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CAAGGTATGGCTGAAGACGAAG-3' ¨³ REF- BamHI 5'-GCAGGCGGATCCTTAATTCTCTCCAAAACA
C-3' š¸É ¤¸‹»—˜´— °ŠÁ°œÅŽ¤r˜´—‹ÎµÁ¡µ³ BsaI ¨³ BamHI (˜ÎµÂ®œn Šš¸É¸—ÁoœÄ˜o ‡º°‹»—˜´— °ŠÁ°œÅŽ¤r) (£µ¡š¸É 1) ×¥
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GoTaq® DNA Polymerase (Promega, Madison, WI ) ž¦´ž¦·¤µ˜¦»—šoµ¥ 25 Pl š¸É°»–®£¼¤· 94 qC 2 œµš¸ ‹Îµœªœ
1 ¦° ˜µ¤—o ª¥ 35 ¦° š¸É 94 qC 30 ª·œµš¸, 60 qC 30 ª·œµš¸, 72 qC 30 ª·œµš¸ ¨³ 72 qC 15 œµš¸ œÎµ PCR
product š¸Éŗo¤µšÎ µ°·Á¨‡Ãš¦Ã¢¦¸ Ž· ĜÁ‹¨°³„µÃ¦ ‡ªµ¤Á o¤ oœ 2% ‹µ„œ´ÊœÂ¥„ cDNA °Š¥¸œ REF °°„‹µ„
Á‹¨°³„µÃ¦Ã—¥Äo QIAEX II (QIAGEN GmbH)
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ž’· ¸ªœ³ ampicillin ‹µ„¦· ¬´š LifeSensor (LifeSensors, Malvern, PA) (£µ¡š¸É 2) ¨³ cDNA °Š¥¸œ REF —oª¥
Á°œÅŽ¤r˜´—‹ÎµÁ¡µ³ BsaI ¨³ BamHI (New England Biolabs Ltd.) ˜µ¤ª·›¸„µ¦ °Š¦· ¬´šŸ¼oŸ¨·˜ ‹µ„œ´ÊœšÎµ
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Á°œÅŽ¤r˜´—‹ÎµÁ¡µ³°°„‹µ„Á‹¨°³„µÃ¦ ץčo QIAEX II (QIAGEN GmbH)
‹µ„œ´Êœ‹¹ŠÁºÉ°¤ cDNA °Š¥¸œ REF „´  pSUMO vector nªœŸ¤ž¦³„°—o ª¥ 1 Pg cDNA °Š REF,
0.5 Pg pSUMO vector, 10X T4 DNA ligase buffer ¨³ 1U T4 DNA ligase (New England Biolabs Ltd.) š¸É°»–®£¼¤·
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