Download - 식품의약품안전청 _KJYDGP001
식품의약품안전청 _KJYDGP001 2007. 11. 13
1. DNA Methylation by EZ DNA Methylateion-Gold Kit (ZYMO RESEARCH)
1-2. The Sample list
1-1. The Method of Methylation
Control DNA_F1, Treated DNA_F1 두 개의 샘플에 대해서 EZ DNA Methylateion-Gold Kit (ZYMO RESEARCH) 을 사용하여 methylation 시킨 뒤 PCR 실험을 수행하였고 ,Negative control 로 methylation 처리를 하지 않은 원상태의 Control DNA_F1, Treated DNA_F1 또한 PCR 실험을 수행하였다
Sample Name
1 Control DNA_F1-Mehtylated
2 Treated DNA_F1-Mehtylated
3 Control DNA_F1-unmehtylated
4 Treated DNA_F1-unmehtylated
식품의약품안전청 _KJYDGP001 2007. 11. 13
1-3. Primer list
1. DNA Methylation by EZ DNA Methylateion-Gold Kit (ZYMO RESEARCH)
Primer PCR product size(bp)
1 Cdkn1c-F1-left/right 4282 Cdkn1c-F2-left/right 4293 Mrps18b_1-F1-left/right 2204 Mrps18b_2-F1-left/right 4885 Mrps18b_2-F2-left/right 4116 Mrps18b_3-F1-left/right 6007 Mrps18b_3-F2-left/right 4278 Tmem56_1-F1-left/right 4539 Vasp_1-F1-left/right 30610 Vasp_1-F4-left/right 41011 Vasp_2-F1-left/right 29812 Pctk1-F1-left/right 71913 Pctk1-F5-left/right 77614 Auts2-F1-left/right 51615 Auts2-F2-left/right 86016 Spata3-F1-left/right 32217 Piwil2-F1-left/right 394
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2. The Result of Secondary PCR
2-1. PCR reaction combination
식품의약품안전청 _KJYDGP001
2-2. PCR Condition
Temp. Time CyclesFinal
Reaction vol.
95 ℃ 5 min 1
30ul
Ex-Taq
94 ℃ 30 sec
3065 ℃ 30 sec
72 ℃ 1 min
72℃ 5 min 1
4℃ Storage 1
PCR 수행 시 Control DNA _F1-Methylated, Treated DNA_F1-Methylated, Control DNA_F1-unmethylated,Treated DNA_F1-unmethylated 4 개의 DNA 샘플과 17 개의 primer set 을 가지고 primary PCR 수행 후 여기서얻은 PCR product DNA 를 template 로 하여 secondary PCR 을 수행하여 결과를 얻었다
Control DNA Treated DNA Control DNA Treated DNAMrps18b_1-F1-L/R 1 2 3 4Mrps18b_2-F1-L/R 5 6 7 8Mrps18b_2-F2-L/R 9 10 11 12Mrps18b_3-F1-L/R 13 14 15 16Mrps18b_3-F2-L/R 17 18 19 20Tmem56_1-F1-L/R 21 22 23 24
Vasp_1-F1-L/R 25 26 27 28Vasp_1-F4-L/R 29 30 31 32Vasp_2-F1-L/R 33 34 35 36
Pctk1-F1-L/R 37 38 39 40Pctk1-F5-L/R 41 42 43 44Auts2-F1-L/R 45 46 47 48Auts2-F2-L/R 49 50 51 52
Spata3-F1-L/R 53 54 55 56Piwil2-F1-L/R 57 58 59 60
Cdkn1c-F1-L/R 61 62 63 64Cdkn1c-F2-L/R 65 66 67 68
Methylated Unmethylated
1
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2-3. PCR Result
2% 0..5X TAE gel
식품의약품안전청 _KJYDGP001
17
41
20
25
53 54
2. The Result of Secondary PCR
2007. 11. 13
2. The Result of PCR
식품의약품안전청 _KJYDGP001
41
1753 54
1kb Ladder
2-4. PCR Result
400bp300bp
800bp
2% 0..5X TAE gel
2% 0..5X TAE gel 2% 0..5X TAE gel
Control DNA Treated DNAMrps18b_3-F2-L/R 17 427
Pctk1-F5-L/R 41 776Spata3-F1-L/R 53 54 322
Methylated PCR productsize (bp)