lab meeting 2014.02 .24
DESCRIPTION
Lab meeting 2014.02 .24. Noh ji eun. Calreticulin. PCR purification - AccuPrep PCR purifiction kit -Components. Calreticulin. Big dye reaction . Calreticulin. Big dye reaction 96 ℃ – 96 ℃ – 50 ℃ – 60 ℃ – 22 ℃ - PowerPoint PPT PresentationTRANSCRIPT
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Lab meeting2014.02.24
Noh ji eun
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Calreticulin
• PCR purification
-AccuPrep PCR purifiction kit
-Components
Buffer 1 (PCR Binding buffer) 250 ㎕
Buffer 2 (Washing buffer) 500 ㎕
Buffer 3 (Elution buffer) 30 ㎕
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Calreticulin
• Big dye reaction
<Big dye mixture>5xbuffer 3.5 ㎕Big dye 1 ㎕F Primer (CALR Exon9 ,5pmol)
1 ㎕
D.W 2.5 ㎕PCR product 2 ㎕Total volume 10㎕
5xbuffer 3.5 ㎕Big dye 1 ㎕R Primer (CALR Exon9 ,5pmol)
1 ㎕
D.W 2.5 ㎕PCR product 2 ㎕Total volume 10㎕
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Calreticulin
• Big dye reaction
<Big dye condition>
96 – 96 – 50 – 60 – 22 ℃ ℃ ℃ ℃ ℃ 1min 10sec 5sec 4min ∞
└ 25cycle ┘
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Calreticulin
• Big dye purification
<Big dye purification mixture>
그 외 필요한 시약 -70%ethanol 350 ㎕ -Hi-Di-Formamide 15 ㎕
Big dye reaction PCR product 10 ㎕3M Sodium acetate(pH4.6) 2 ㎕95% ethanol 50 ㎕
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Calreticulin
• Sequencing Result
-CALR1,2,3,5,6,7,8,9,10,11,12,14,15
:mutation 없음 .
-CALR4,13
:Sequencing Result 나오지않음 .
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Calreticulin
• PCR reactionPCR mixture
10xF-Taq buffer 5 ㎕dNTP 1 ㎕Primer Fam (Exon 9 ,20pmol) 2 ㎕Primer R (Exon 9 ,20pmol) 2 ㎕F-Taq 0.3 ㎕DNA(Sample) 100ng/ ㎕당 1.5 ㎕D.W 38.2 ㎕Total volume 50 ㎕
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Calreticulin
• PCR Condition95 ℃ 5min
10Cycle
1 per cycle ℃ →
94 ℃ 30sec ℃
67 ℃ 30sec ℃
72 ℃ 30sec ℃
29Cycle 94 ℃ 30sec ℃
57 ℃ 30sec ℃
72 ℃ 30sec ℃
72 ℃ 10min ℃
18 ℃ ∞
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Calreticulin
• PCR Result → Electrophoresis
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
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Calreticulin
• Gene scan
• Gene scan PCR condition
: 95 – 4℃ ℃ 5min ∞
PCR product(20 배 diltion) 1 ㎕Hi-Di-Formamide 13.7 ㎕Rox standard size ladder 0.3 ㎕Total volume 15㎕
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Calreticulin
• Gene scan result
:CALR 1,2,3,6,8,9,10,11,12,14-mutation 없음 CALR 5,7-mutation 있음
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Calreticulin
• Sample: MPN 환자에서 2011 년도 2 명 , 2012 년도 45 명 JAK2 (-) 환자 .
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• PCR Result → Electrophoresis
Calreticulin
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30
31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47
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Thank You