2）培養細胞からの RNA の回収と cDNA の合成操作
Contents
1. 1 RNA RNA 2008 2 1 2005 582. 1 cDNA RNA RNA cDNA PCR DNA 1 2 SuperScript II Reverse Transcriptase RT Invitrogen cDNA dT 50uM 50 250ng 10mM dNTP RNase free 1 1 5mL cDNA 1luL 10mM dNTP luL 1 Sus total RNA
3. 15 2mL 4 500uL Buffer RPE 8 000xg 15 2mL 5 500uL Buffer RPE 8 000xg 6 RNeasy 1 5mL 30 SO0uL RNase free 8 000xg 1 7 80 C 1 RNA RNA NM 3 4 x 10 2 80 C 80C 3 B
4. 10 10 total RNA 100ug 1 5 2 0mL Buffer RLT Buffer RW1 Buffer RPE RNase free 14 3M B B ME 70 100 RNase free 2 Buffer RLT 1mL 10uL B ME 3 1 Buffer RPE 4 100 20 25 C 2 3 2 Total RNA 1 53mL 300xg 5 Buffer RLT
5. 2 RNA total RNA 1 20 21G RNeasy Mini Kit QIAGEN Co 20
6. RNase RNase free RNase 0 1N NaOH 1mM EDTA RNase RNase free RNase free RNA RNase free RNase DEPC RNase RNase free RNA RNA
7. AGPC Acid Guanidium Phenol Chloroform RNase RNA AGPC 49 20 7 1 2 TRIzol Reagent Invitrogen ISOGEN RNAiso Plus easy BLUE
8. RNA RNA 1 5mL 15mL 50mL 15mL 50mL Total RNA RNA RNA RNA 1 DNA RNA DNA RNA
9. 2 RNA 1 2 3 4 2 3 4 1 8 5uL 2 5uL 10xMOPS 2 0uL 0 4mgml 1 0uL RNA 10ng RNase free 4uL 65 C 10 10x 2 0uL RNase free 1xMOPS 1 2 2 1 RNA SE S0V 1 5 2 BPB 8 UV 28S 18S RNA 28S 18S 2 1 RNA 55
10. 20 2 RNA cDNA dl NN mRNA
11. 1 2 RNase free 25 500ng uL RNA 1 12 3 4 5 6 0 28S RIN 9 8 18S 59 3 Agilent 2100 Electropherosram RNA 28s 18s RNA Integrity Number RIN RIN RNA 1 10 10 56 20
12. 20 3 Agilent 2100 Agilent Technologies Agilent 2100 RNA DNA RNA 1 Agilent 2100 Agilent Technologies 2 Agilent RNA 6000 Nano Kit Agilent Technologies RNase free pl
13. 1 TE pH A260 A280 2 100 200uL 2 RNA E RNA QrL 1 Mupid UV 2
14. 2 75 RNase free 1l 1 2 3 4 5 3 1 2 PBS 2 TRIzol Reagent 10cm TRIzol Reagent 1mL 35mm 10cm 1 3mL PBS 2 5 10x10 TRIzol Reagent 1mL 1 5mL ul 2 Bn as 3 1 15 30 C TRIzol Reagent lmL 0 2mL
15. 1mL 75 7 500xg 4 C 5 RNA 10 13 10 100uL RNase free RNA 80 C 1l 80 C 1 4 RNA 72 2 8 C 1 20 C 1 2 10 10 4 RNA 5 RNA
16. 10xMOPS 0 2M MOPS 50mM 10mMEDTA pH7 0 NaOH pH 54 20 36 38 209C 10x 50 10mM BEDTA 0 05 BPB 0 05 RNase free 0 4mg ml 1 1 RNase free 84 5mL 1 2g 2 60 C 10xMOPS 10mL 5 5mL 3
17. 15 2 3 12 000xg 4 C 10 1 RNA 2 1 5mL 1 TRIzol Reagent 1mL 0 5mL lt em 1 TRIzol 50 3 4 1 2 3 4 20 7 10 12 000xg 4 C 10 RNA 3
18. 13uL RNase free 2 65 C 5 SuperScript IT RT 5xFirst Strand Buffer 4uL 0 1M DTT 2uL SuperScript IT RT 1uL 25 C 50 C 30 60 70 C 13 20 C CO Q Q PR 1 2 1 0 2mL 0 5mL PCR 2 2 55 C 3 SuperScript II RT cDNA RNase H RNA cDNA RNA 2
19. TE 53 20 4 260nm 280nm 1 RNA RNA 260nm A260 1 0 04uguL A260 0 04 uguL A260 0 3 100 RNA ug uL 0 3 x 0 04 x 100 1 2 2 RNA A260 A280 1 9 2 1
20. 2 cDNA PCR 5 20 1 PCR 1kb 37 C 20 RNase H RNA RNA RNase RNase RNase free RNA RNA RNase
21. Buffer RLT 1 30 5 j gt 1 Buffer RLT Buffer RLT uL cm 350 lt 5x10 lt 6 600 Sx10 107 6 10 Buffer RLT 70 700nL 2mL RNeasy 8 000xg 15 2mL 700uL 3 700uL Buffer RW1 8 000xg 5 20
22. uffer RPE RNA Buffer RPE RNase free 2mL 1 RNA 1 1 TE 10mM Tris HCI 1mM EDTA pHS8 0 1 260nm 260nm 280nm 2 1 5mL TE RNA 30 200 0 1 1 0 3
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