Lab meeting2014.02.24

Noh ji eun

Calreticulin

• PCR purification

-AccuPrep PCR purifiction kit

-Components

Buffer 1 (PCR Binding buffer) 250 ㎕

Buffer 2 (Washing buffer) 500 ㎕

Buffer 3 (Elution buffer) 30 ㎕

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㎕

Calreticulin

• Big dye reaction

<Big dye condition>

96 – 96 – 50 – 60 – 22 ℃ ℃ ℃ ℃ ℃ 1min 10sec 5sec 4min ∞

└ 25cycle ┘

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 ㎕

Calreticulin

• Sequencing Result

-CALR1,2,3,5,6,7,8,9,10,11,12,14,15

:mutation 없음 .

-CALR4,13

:Sequencing Result 나오지않음 .

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 ㎕

Calreticulin

• PCR Condition95 ℃ 5min

10Cycle

1 per cycle ℃ →

94 ℃ 30sec ℃

67 ℃ 30sec ℃

72 ℃ 30sec ℃

29Cycle 94 ℃ 30sec ℃

57 ℃ 30sec ℃

72 ℃ 30sec ℃

72 ℃ 10min ℃

18 ℃ ∞

Calreticulin

• PCR Result → Electrophoresis

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

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㎕

Calreticulin

• Gene scan result

:CALR 1,2,3,6,8,9,10,11,12,14-mutation 없음 CALR 5,7-mutation 있음

Calreticulin

• Sample: MPN 환자에서 2011 년도 2 명 , 2012 년도 45 명 JAK2 (-) 환자 .

• 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

Thank You