Summer Research

1
Summer Research

• ????
• ?????04?
• ???
• 891645

2
Introduction

• 2002?????????????????-???????????????????????????-
  ???????????????,?????????????????????????????(Sup
  pressor Subtractive hybridization,SSH)????????????
  ????
• ????SSH????????cDNA library????????????gene
  cloned,??insert??????,?????,????????
• ???cloned???gene GMF-ß?s100?
• ????????(rostral ventrolateral medulla(RVLM
  ))cDNA

3
????

• (1)?RVLM?cDNA ,???clone gene?primer(fr),
  ???Taq polymerase ,run PCR?
• (2)Run electrophoresis,check?????
• (3)??kit?purify PCR product?
• (4)?????????DNA???????
• (5)Run electrophoresis,?marker????????DNA???
• (6)???????????-200C ???

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????

• (7)??????transformation?, ????????agar plate(with
  amp w/o amp) ??????competent cell (?? e.coli
  JM-109) ?
• (8)   Ligation ????PCR?????????pGEM T easy
  vector??ligation ?
• (9)Transformation
• (10)?plate??? (?????)????,????LB
  medium????,??16-20hr?
• (11)??,?plasmid ,?EcoR I?3hr,??electrophoresis????
  ???insert?

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????

• (12)??insert?????,????????????
• (13)?kit ?plasmid ,?????plasmid??,
  ???check???or???
• (14)?????insert??restriction enzyme???vector????,?
  ????restriction enzyme??, ?????????????????GMF-B?s
  100????,??????enzyme??????????????????????(???????
  ??????) ?

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????
7
????

• 1. GMF-ß
• (1)   PCR????????????

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????

• (2)   Transformation????, ??????competent
  cell?????, ??ligation????? ?
• (3)   restriction enzyme????

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????

• ???????insert , ???????insert?????,???plasmid ?
• (4)???
• (5)??????recombined plasmid?????

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???
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????

• 2. s100
• (1)   PCR????????????

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????

• (2)Transformation????, ??????competent cell?????,
  ??ligation??????
• (3)restriction enzyme????

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????

• ????!!???????????,???plasmid????PCR
  ,???insert?????(????) ?

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????

• ???no.3???, no.1???band????,????2.4.5.6???????????
  no.1????????PCR?,??????,??primer??????????,???????
  ?????????
• ligation?????recombined DNA
  ,?transformation?????????S100 ????insert???????,?
  ???????no9-12 ?

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????

• ?plasmid???,????no.2?????plasmid
  PCR????,?????????????insert????DNA??

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????

• ??????plasmid?kit??, ????????, ??????s100???
  --????????? ?

17
??

• ??????????????,????????
• ??????????????????,????
• ???????,???????????????
• ????????????,??????????
• ??PCR.?plasmid.??. ???????????
• ??,??????,??????????????