cDNA Library Construction Kit
Contents
1. 16 1 TafdR4 Tel 077 543 6116 Fax 077 543 1977 http www takara bio coJp v201308Da2. 3 4 1 5 ml FALCON 2 700Xg 5 1 5 ml FALCON Aor TE A 7 1 2 3 4 5 6 7 8 9 10 11
3. 2 AlkPhos Direct Labelling Detection System GE http wwwr takara bio coJp 15 6136 X 1 Gubler Hoffman J 1983 25 263 2 1994 2 p79 94 Wokdnar Filipowicz et al 1984 Proc 5 USA 81 2295 Howells et al 1984 Natl 5 USA 81 7651 Schneider et al 1984 Nature 311 675 Leis et g 1973 Proc Acad Sci USA 70 466 Okayama Berg 1982 Mol Cell Biol 2 161 Kobori Ikeda Kumegawa Kawashima 1998 Ge es Ce 75 3 459 475 3 4 5 6 7 8 PrimeScript Reverse Transcriptase 2680A B C Recombinant RNase Inhibitor 2313A B Polymerase
4. cDNA polyA cDNA cDNA cDNA vitro transcription in vitro translation cDNA Library Construction Kit polyA cDNA cDNA pAP3neo SV40 cDNA Gubler Hoffman 2
5. 1 Control RNA control pSP Tet3 polyA 1st Strand 2nd Strand cDNA 2 polyAT cDNA polyA RNA RNA cDNA cDNA cDNA 260 nm 280 nm A2eo A2go 1 8 2 1 RNA 3 RNase RNase
6. VIII 3 2 PrimeScript Double Strand cDNA Synthesis Kit 6111A 3 Gen 9094 4 EcoRI or 5 Control RNA SP6 promoter pBR322 1 4 kb pSP Tet3 SP6 Polymerase vitro transcription 1 Control Control 30 polyA tail 1 4 kb cDNA cDNA full length 6
7. 4 SOC 960 5 FALCON 37 1 6 5 1 ul 10 ul LB Amp 4 2 7 37 8 9 22 T7 promoter primer 23 promoter primer for 3 PCR 1 CDNA E coli HST08 Premium Electro Cells 9028 Stellar Electrocompetent Cells 636765 DNA
8. DNA 80 1 AGPC RNAiso Plus 9108 9109 NucleoSpin 740955 10 50 250 2 polyA polyA dT Cellulose Poly Sepharose RNA O 9o7ex 9730 lt 5upe gt O 9o7ex 730 lt Super gt mRNA Purifcation polyA 3 RNA cDNA polyA cDNA RNA
9. 7 Clontech Laboratories DNA E HST08 Premium Electro Cells 9028 Clontech 0 Stellar Electrocompetent Cells 636765 http wwwr takara bio coJp 5 6136 lt gt HST08 Premium Electro Cells 9028 Stellar Electrocompetent Cells 636765 Clontech 25 24 1 v v v 24 1 v v TE 10 mM Tris HCI 1 mM EDTA pH8 0 DNA 1 0 1 u g ml
10. 4 ES 2z http www takara bio co jp 13 6136 1 Oligo dT 18 Anchor Primer 5 GA 10 CTCGAGCGGCCGC T 1gV 3 V AorCorG Anchor Primer Not Xho 1 pAP3neo EcoR 5ma Adaptor 5 3 GGGCCC 5 7 promoter primer 5 3 3 promoter primer for pAP3neo 5 3 pAP3neo 2 pAP3neo SV40 promoter Poly A signal Splice signal Poly A signal pAP3neo Predigested Vector EcoR 1 Not
11. Directional cloning 1 1 PrimeScript RTase 18 Anchor Primer 1 3 5 1st Strand cDNA 1st Strand cDNA 5 methyl 2 coli 6 mRNA 1st Strand cDNA RNA Polymerase E coli Ligase RNA 2nd Strand cDNA 1 7 3 T4 Polymerase 4 Mor 5 DNA 6 pAP3neo 8 Directional cloning 7 8 http wwwr takara bio coJp 3 6136 T 18 _ Ao _ 1 PrimeScript RTase 151 Oligo dT 1s Anchor Primer
12. 20 ul 11 10 XT4DNA Ligase Buffer 2 13 EcoR Sma Adapter 3 5 ul 12 T4DNA Ligase 2 2 8C 3 70 30 5 V 4 Motl 1 20 ul 50 14 orISupplement 27 15 3 ul 2 37 3 http www takara bio co jp 9 6136 5 400 bp DNA 1 2 3 4 5 6 7 8 9 10 1 2 10 1 ml TE
13. V 7 15 cm LB Amp 100 ug ml ampicillin 5 37 6 8 LB Amp 100 ug ml ampicillin 4 1 2 0 5 NaOH 0 5 30 2 1 1 Tris HCI pH7 6
14. 1 1 2 ug 65 10 2 ribosomal 285 185 2 1 ribo somal RNase 28S DNA Recombinant DNase RNase free 2270A B cDNA 2100 Agilent RNA6000 Nano LabChip 2 RNA polyAT A2eo A2gso 1 7 1 8 2 1 http wwwr ta
15. 16 C 2 70 10 5 10 4 Polymerase 4 37 10 2nd Strand cDNA 150 25 24 1 150 ul 5 10 15 000 rpm 5 2 24 1 150 ul 5 10 15 000 rpm 5 2 1 10 18 3M Sodium Acetate pH5 2 17 GenTLE Precipitation Car rier 4 2 2 5 15 000 rpm 30 70 12 5 ul 20 RN
16. 30 2 1 1 MTris HCI pH7 6 1 5 NaCI 30 1 Tris HCI pH7 6 1 5 NaCl 1 Tris HCI pH7 6 1 5 NaCl 80 2 DNA UV K 1
17. EcoR Predigested Vector EcoR T7 Splice signal transcription SV40 promoter GenBank Accession No AB 003468 3 Ao7 Xho 1 V 3 1 3 Xho 1 1094AH H Buffer 50 ull 37 3 http www takara bio co jp 14 6136 IX 1 1 2 3 4 6 coli HST08 Premium Electro Cells V 7 15 cm LB Amp 1
18. 70 15 ul 20 RNase free 20 100 TE http wwwr takara bio coJp 10 6136 6 1 11110 T4 DNA Ligase Buffer 2 cDNA 15 ul 19 Predigested Vector 12 4 Ligase 2 2 12C 3 TE 80 ul 25 24 1 100 ul 5 10 4 15 000 rpm 5 2 5 24 1 100 ul 5 10 6 15 000 rpm
19. 61 36 cDNA Library Construction Kit vV201308Da I cDNA 1 2 3 RNA V V 1 1st Strand cDNA V 2 2nd Strand cDNA V 3 V 4 Motl V 5 DNA V 6 V 7 1 2 pAP3neo 3 IX 1 IX 2 X http wwwr takara bio coJp 2 6136 1 polyA cDNA
20. DEPC 37 12 DEPC RNA lt RNase RNA 2 0 1 DEPC http wwwr takara bio coJp 6 6136 3 RNA RNA RNA cDNA
21. LB LB Amp 100 ug mD lt gt 8 12 16 37 42 70 FALCON BD Code No 352059 17 Dr GenTLE Precipitation Carrier 18 3M Sodium Acetate pH5 2 4 20 IV cDNA 1 RNase 160 2 0 1
22. 50 ul 4 2 4 4 3 10 0 1 0 50 Primer 05 l 2 5 pmol 95 1 40 VIII 94 30 55 30 30 72 3 72 102 http wwwr takara bio coJp 12 6136 VI Chicken polyA 5 ug cDNA 16 PCR DNA 1 1 2 2 1 Lane 1 AA EcoT141digest 2 pHY Marker
23. co7 Ligase Mixture 10 ul 7 20 U 10 ul 8 2nd Strand dNTP Mixture 23 ul 9 5X2nd Strand Synthesis Buffer 2 150 10 T4 DNA Polymerase 1 U ul 20 11 10 Xx T4 DNA Ligase Buffer 20 12 T4DNA Ligase 350 U pD 20 13 EcoR Sma Adaptor 0 4 ug ul 18 ul 14 1 135 ul 15 or 50 0 15 ul 16 tRNA 10 9 ul 5 17 Dr GenTLE Precipitation Carrier 3 60 4 18 3M Sodium Acetate pH5 2 1 ml 4 19 pAP3neo Predigested Vector 100 ng ul 4 5 ul 20 RNase free H2O 640 wl 21 Control 1 gt 5 ul 22 7 promoter primer 6 5 pmol ul 20 23 promoter primer for pAP3neo 6 5 pmol 1 20 CHROMA SPIN 1000 DEPC H20 Columns 5 4 1 Oligo dT 1g Anchor Primer Not Xho Not Xho 1 EcoR Xho cDNA cDNA Xho
24. co 2130A B 4 Polymerase 2040A B T4 Ligase 2011 4 Polynucleotide Kinase 20215 pHY Marker 3404A B RNAiso Plus 9108 9109 NucleoSpin 740955 10 50 250 Oligotex 730 lt 5 gt W9021A B Oligotex 9730 lt Super gt mRNA Purification Kit From Total 9086 Gen 9094 coli 8 Premium Electro Cells 9028 E coli HST02 Electro Cells 9026 Stellar Electrocompetent Cells 636765 CHROMA SPIN 1000 DEPC H20O Columns 636093 636094
25. 5 methyl Not Xho 1 AAAAAAAAAAAAAAAAAAAAAAAAA AG io methy dCTP Polymerase E coli RNaseH DNA Ligase Mixture 2nd 4 polymerase AAAAAAAAAAAAAAAAAAGCGGCCGC_ TOio TTTTTTTTTTTTTTTTTTCGCCGGCG _ AG 1o M EcoR1Adaptor 4 Ligase EcoR 1 EcoRI Not EcoR M EcoR I Not pAP3neo Predigested co7 HST08 Premium Electro Cells coli HST02 Electro Cells Stellar Electrocompetent Cells cDNA 1 cDNA http www takara bio coJp 4 6136 5 1 PrimeScript RTase 200 U ub 5 2 RNase Inhibitor 40 U uD 5 3 Oligo dT 18 Anchor Primer 1 ug uD 1 10 ul 4 5 1st Strand Synthesis Buffer 2 20 ul 5 1st Strand dNTP Mixture 6 co RNase
26. oz 50 ul TE 40 ul 16 tRNA 1 ul B 1 A 10 ul 700Xg 5 100 ul 1 5 ml 25 24 1 100 5 10 15 000 rpm 5 2 24 1 100 ul 5 10 15 000 rpm 5 2 1 10 18 3M Sodium Acetate pH5 2 17 GenTLE Precipitation Carrier 4 2 2 5 15 000 rpm 30
27. 00 ug ml ampicillin 5 37 LB Amp 100 ug mlampicillin 2 1 LB Amp 100 ug mlampicilin 2 ml 1 20 80 2 2 IX 2 1 2 3 4 5 6 7 8 9 10 11 12 co HST08 Premium Electro Cells
28. 5 2 7 1710 20 18 Sodium Acetate pH5 2 17 GenTLE Precipitation Carrier4 2 2 5 8 15 000 rpm 30 9 70 10 20 ul TE 11 20 http wwwr takara bio coJp 11 6136 V 7 1 Stellar Electrocompetent Cells 70 2 0 5 1 0 40 3 0 1 cm 1 05 1 0 Gene Pulser Bio Rad Stellar Electrocompetent Cells 1 5 200 25 uF
29. ase free 20 http wwwr takara bio coJp 8 6136 polyA 2nd cDNA polyAT RNA cDNA cDNA RNA cDNA RNA 1 4 5 ug Oligo dT 18g Anchor Primer 2nd Strand cDNA M1 1 M2 Lane 1 2ndStrand cDNA 1 AcoT141digest 2 pHY Marker 2nd Strand cDNA V 3 1 cDNA 12 5
30. kara bio coJp 7 6136 V V 1 1st Strand cDNA 1 4 6 3 Oligo dT 18 Anchor Primer 2 5 1stStrand dNTP Mixture 1 2 ul SNA polyAT 5 9 20 RNase free H2O up to 10 65 5 20 ul RNA Primer mixture 10 ul 4 5X1stStrand Synthesis Buffer 4 ul 2 RNase Inhibitor 1 PrimeScript RTase 20 RNase free H2O 4 ul 42 1 2 Not X7o1l V 2 2nd Strand cDNA 1 10 11 12 13 1st Strand cDNA 20 ul 146 9 5 2nd Strand Synthesis Buffer 30 8 2nd Strand dNTP Mixture 4 5 ul 20 RNase free H2O 87 5 ul 6 Eco 7RNaseH E coli Ligase Mixture 2 7 Polymerase 2
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