1. Lab meeting 2014.02.24 Noh ji eun

2. Calreticulin PCR purification -AccuPrep PCR purifiction kit -Components Buffer 1 (PCR Binding buffer) 250 ㎕ Buffer 2 (Washing buffer) 500 ㎕ Buffer 3 (Elution buffer) 30 ㎕

3. Calreticulin Big dye reaction 5xbuffer 3.5 ㎕ Big dye 1㎕1㎕ F Primer (CALR Exon9,5pmol) 1㎕1㎕ D.W 2.5 ㎕ PCR product 2㎕2㎕ Total volume 10 ㎕ 5xbuffer 3.5 ㎕ Big dye 1㎕1㎕ R Primer (CALR Exon9,5pmol) 1㎕1㎕ D.W 2.5 ㎕ PCR product 2㎕2㎕ Total volume 10 ㎕

4. Calreticulin Big dye reaction 96 ℃ – 96 ℃ – 50 ℃ – 60 ℃ – 22 ℃ 1min 10sec 5sec 4min ∞ └ 25cycle ┘

5. Calreticulin Big dye purification 그 외 필요한 시약 -70%ethanol 350 ㎕ -Hi-Di-Formamide 15 ㎕ Big dye reaction PCR product 10 ㎕ 3M Sodium acetate(pH4.6) 2㎕2㎕ 95% ethanol 50 ㎕

6. Calreticulin Sequencing Result -CALR1,2,3,5,6,7,8,9,10,11,12,14,15 :mutation 없음. -CALR4,13 :Sequencing Result 나오지않음.

11. Calreticulin PCR reaction PCR mixture 10xF-Taq buffer 5 ㎕5 ㎕ dNTP 1 ㎕1 ㎕ Primer Fam (Exon 9,20pmol) 2 ㎕2 ㎕ Primer R (Exon 9,20pmol) 2 ㎕2 ㎕ F-Taq 0.3 ㎕ DNA(Sample) 100ng/ ㎕당 1.5 ㎕ D.W 38.2 ㎕ Total volume 50 ㎕

12. Calreticulin PCR Condition 95 ℃ 5min 10Cycle 1 ℃ per cycle → 94 ℃ 30sec ℃ 67 ℃ 30sec ℃ 72 ℃ 30sec ℃ 29Cycle 94 ℃ 30sec ℃ 57 ℃ 30sec ℃ 72 ℃ 30sec ℃ 72 ℃ 10min ℃ 18 ℃ ∞

13. Calreticulin PCR Result → Electrophoresis 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

14. Calreticulin Gene scan Gene scan PCR condition : 95 ℃ – 4 ℃ 5min ∞ PCR product(20 배 diltion)1㎕1㎕ Hi-Di-Formamide 13.7 ㎕ Rox standard size ladder 0.3 ㎕ Total volume 15 ㎕

15. Calreticulin Gene scan result :CALR 1,2,3,6,8,9,10,11,12,14-mutation 없음 CALR 5,7-mutation 있음

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

20. 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

21. Thank You