accura-expRACE KIT
Contents
1. RALE oroduct c T Sequencing primer 5 RACE primer C m 7 c 5 3 RACE RACE mTfre 5 RACE 3 RACE ds cDNA RACE m 7 c RT PCR PCR RACE PCR PCR RT PCR RT PCR 5 10 Template switching 1 RACE PCR RACE PCR2. DNA GC 24 mer cDNA PCR 24 34 bp Terminal Inverted Repeat cDNA A RACE PCR RT PCR RACE PCR PCR PCR PCR Tm 60C 65C Thermal Cycling DNA Polymerase RT PCR 5 10 Template switching 5 5
3. RACE RT PCR Template switching cDNA 1 5 RACE cDNA Template switching 3 RACE 3 RACE PCR RT PCR 1 RT PCR M AHindlll digestion 2 11 5 RACE PCR 2 5 1 RACE 6 cDNA 1 10 8 7 8 cDNA 1 2 1 5 4 9 10 11 cDNA 1 2 4 RACE Tm RACE PCR
4. RACE m Tfrc cDNA 4920 bp gt 3 RACE primer 5 RACE primer 2109 2131 4358 4380 total RNA poly A RNA ds cDNA total RNA poly A RNA VI A ds cDNA Tfrc 8 actin D RACE PCR RT PCR ds cDNA X 1 IV 20 1 250
5. DNA DNA 1st cycle denature gene specific primer synthesis 3 end synthesis by DM 1 m template switching of ia A 9 Taq DNA polymerase 3 end 5 7j P EM a 2nd cycle sequences complementary template DNA to a gene specific primer P denature annealing synthesis 2 RACE DNA 5 DNA 5 Template switching RACE cDNA Terminal Inverted Repeat 24 34 bp 5 68C ds cDNA 40 bp
6. accura expRACE KIT amp kjuro ikspr 1 s El Plain Institute Revolutionary Tools for Continual Discovery Code No EPIOO Lot No 0001 Storage 20 C 5 cDNA RACE PCR DNA poly A RNA A First Strand cDNA 1 100 ul 5X RTase Reverse Transcriptase Buffer 250 mM Tris HCl pH 8 3 375 mM KCI 15 mM MgCL 2 10 ul Oligo dT Primer 10 uM 3 5 5 ul M MLV Moloney Murine Leukemia Virus Reverse Transcriptase 50 U ul B Second Strand cDNA 4 200 ul 5XSecond Strand Synthesis Buffer 100 mM Tris HCI pH 7 5 500 mM KCI 2b mM MgCL 50 mM NH4 SO Ammonium Sulfate 5 mM Dithiothreitol DTT 0 25 mg ml
7. Il 1 DNA Polymerase PrimeSTAR TaKaRa KOD TOYOBO Pfu Thermo Fisher Scientific Phusion New England Biolabs etc 2 Phenol TE pH 8 0 3 99 596 Ethanol 4 75 Ethanol 5 TE pH 8 0 Ill First Strand cDNA M MLV RTase Moloney Murine Leukemia Virus Reverse Transcriptase cDNA First Strand cDNA GC RNA 42C First Strand cDNA cDNA CDNA First Strand cDNA 5X RTase Buffer 2 ul 10 mM dNTP 1 ul 10 uM Oligo dT l ul poly A RNA 1 ug 1 5 ul M MLV RTase 50 U ul l ul IREK 4 0 ul Total volume 10 ul 1 poly A RNA Oligo dT 0 5 ml
8. D Genomic DNA RACE cDNA poly A RNA DNA poly A RNA total RNA RNase DNase DNA poly A RNA total RNA DNase RNase DNase poly A RNA total RNA DNase RNA total RNA RACE
9. T111 0033 1 12 2 TEL 03 6231 6605 Email contact elplain com 12
10. DNA 6 7 8 DNA 5 DNA DNA 5 Template switching 3 DNA 1 cDNA cDNA 3 gt Terminal Inverted Repeat 9 10 11 12 13 2 ds cDNA PCR 5 RACE 3 RACE 4 304 350 U S patent No 7 504 240
11. poly A RNA poly A RNA total RNA Off 1 poly A RNA 14 poly A RNA 1 g poly A RNA cDNA TE pH8 0 ds cDNA RACE poly A RNA 15A cDNA B RACE RACE PCR RACE 1 ds cDNA 1 2 1 5 1 10 2 ds cDNA 2 3 3 RAC
12. VIII Conclusion accura expRACE KIT RACE cDNA IX 1 Okayama H amp Berg P 1982 High efficiency cloning of full length cDNA Mol Cell Biol 2 161 170 2 Gubler U amp Hoffman B J 1983 A simple and very effective method for generating cDNA libraries Gene 25 263 269 3 Frohman M A Dush M K amp Martin G R 1988 Rapid production of full length cDNAs from rare transcripts amplification using a single gene specific oligonucleotide primer Proc Natl Acad Sci USA 85 8998 9002 4 Chenchik A Diachenko L Moqadam F Tarabykin V Lukyanov S amp Siebert P D 1996 Full length cDNA cloning and determination of mRNA 5 and 3 ends by amplification of adaptor ligated cDNA BioTechniques 21 526 534 5 Hirano M 2004 RACE using only a gene specific primer application of a template switching model Mol Biotechnol 27 179 186 6 Levene S D amp Crothers D M 1986 Ring closure probabilities for DNA fragments by Monte Carlo simulation J Mol Biol 189 61 7
13. mTfrc RACE 5 RACE PCR RT PCR DNA KOD Plus Neo TOYOBO Thermal Cycling RACE PCR 26 ul 1OX PCR Buffer 5 ul Tfrc 5 RACE Primer 10 pmol ul 5 ul 2 mM dNTP 5 ul 25 mM MgSO 3 ul ds cDNA 5 ul KOD Plus Neo 1 U ul 1 ul Total volume 50 ul RT PCR TALES LZK 28 ul 10X PCR Buffer 5 ul Tfrc 3 RACE Primer 10 pmol ul forward 1 5 ul Tfrc 5 RACE Primer 10 pmol ul reverse 1 5 ul 2 mM dNTP 5 ul 25 mM MgSO 3 ul ds cDNA 5 ul KOD Plus Neo 1 U ul 1 ul Total volume 50 ul Thermal Cycling 94 C 2 min 98 C 10 sec 63 C 30 sec 40 cycles 68 C 4 min RACE PCR 10ul KU RT PCR 2ul 1 3 3 pD9 4 4 kb 3 kb 2 4 kb 5 RACE 2 1 1 2 3 kb RT PCR 1 5 RACE 3 RACE 4
14. cDNA ds cDNA DNA cDNA 14 15 DNA cDNA TE pH8 0 RACE PCR RACE PCR 2 3 8 10 4 RACE 3 6 8 PCR VI A ds cDNA poly A RNA ds cDNA total RNA rRNA 288 RNA 18S RNA tRNA 5S rRNA
15. 5X RTase Buffer 10 mM dNTP Mix 2 1 ug poly A RNA 1 5 ul Oligo dT Primer 1 ul 0 5 ml 3 WAPSZK HA c6 ul 4 4C 5 70C 3 6 3 7 4C 8 5X RTase Buffer 2 ul 10 mM dNTP 1 ul M MLV RTase 50 U ul 1 gl Total volume 10 ul 9 10 4C 11 42C 1 12 2 3 13 Second Strand cDNA IV Second
16. opidi 1 2C RACE PCR 1 2 C RACE PCR 1 Wine EUER RACE cDNA Tm RACE E RACE cDNA RACE cDNA DNA QU e cDNA cDNA cDNA RACE
17. RT PCR 1 2 RACE PCR 50 ul PCR TALES ZK 29 ul 1OX PCR Buffer 5 ul Gene specific RACE Primer 10 pmol ul 5 ul 2 mM dNTP 5 ul ds cDNA 5 ul 1 2 ng DNA 1 U ul 1 ul Total volume 50 ul Thermal Cycling 94C 2 min 98C 10 sec Tm C 30 sec 35 40 cycles 68C 4 min DNA PCR 5 10 ul DNA RACE B DNA RACE PCR TA DNA cDNA Direct DNA Sequencing RACE PCR 1
18. VII RACE 5 RACE 3 RACE cDNA PCR RT PCR PCR RACE cDNA H ds cDNA X cDNA 5 1 RACE PCR cDNA 6 in vitro DNA 5 RACE cDNA 7 T4 DNA ds cDNA cDNA cDNA T4 DNA mRNA 16
19. 17 cDNA 18 19 8 RACE Expressed Sequence Tag EST 20 Serial Analysis of Gene Expression SAGE 21 Differential Display 22 RNA cDNA 9 microRNA pri miRNA 23 IncRNA 24 25 cDNA RACE 8 10 DNA insertional mutagenesis DNA DNA DNA DNA 10
20. PldCTP cDNA 19 10 ul TE pH 8 0 1 ul ds cDNA 1 1 ds cDNA ds cDNA poly A RNA 1 ul cDNA 1 1 2 ds cDNA L 7 M AHindlll digestion 1 ug O poly A RNA 0 2 1 ug ds cDNA V Rapid Amplification of cDNA Ends RACE 3 4 RACE 1 cDNA 5 2 p6 2 DNA cos PCR 68C
21. 2 T Cloutier T E amp Widom J 2004 Spontaneous sharp bending of double stranded DNA Molec Cell 14 355 362 8 Du Q Smith C Shiffeldrim N Vologodskaia M amp Vologodskii A 2005 Cyclization of short DNA fragments and bending fluctuations of the double helix Proc Natl Acad Sci USA 102 5397 5402 9 Panganiban A T amp Temin H M 1983 The terminal nucleotides of retrovirus DNA are required for integration but not virus production Nature 306 155 160 10 Wang K amp Pearson G D 1985 Adenovirus sequences required for replication in vivo Nucleic Acids Res 13 5173 5187 11 Olasz F Farkas T Kiss J Arini A amp Arber W 1997 Terminal inverted repeats of insertion sequence IS30 serve as targets for transposition J Bacteriol 1 79 7551 7558 12 Calvi B R Hong T J Findley S D amp Gelbart W M 1991 Evidence for a common evolutionary origin of inverted repeat transposons in Drosophila and plants hobo Activator and Tam3 Cell 66 465 47 1 13 Morgan G T 1995 Identification in the human genome of mobile elements spread by DNA mediated transposition J Mol Biol 254 1 5 14 Ausubel F M Brent R Kingston R E Moore D D Seidman J G Smith J A amp Struhl K Eds 1994 Current Protocols in Molecular Biology John Wiley amp Sons Inc New York 15 Sambrook J Fritsch EF amp Maniatis T 1989 Molecular Cloning A Laboratory Manual 2nd
22. Bovine Serum Albumin BSA 0 75 mM 5f Nicotinamide Adenine Dinucleotide 8 NAD 5 5 5 ul E coli RNase H 1 U ul 6 5 5 ul E coli DNA Ligase b U ul 7 11 ul E coli DNA Polymerase I 12 U ul 8 300 ul EDTA 0 5 M EDTA pH 8 0 C 9 40 ul 10 mM dNTP Mix 10 mM each of dATP dCTP dGTP dT TP 10 500 ul 7 5 M Ammonium Acetate 11 1 7ml WAK Distilled Water Deionized Sterile 12 1 6ml TE pH 8 0 10 mM Tris HCI pH 8 0 1 mM EDTA pH 8 0 D RACE PCR Control 5 and 3 RACE Primers 13 200 ul Mouse Transferrin Receptor 7 o 5 RACE Primer 10 pmol ul 5 TTCTCAGGIGGCAGCTTTGAACT 3 Tm 62 58 C 14 200 ul Mouse Transferrin Receptor 7 o 3 RACE Primer 10 pmol ul 5 CGTGGAGACTACTTICCGTGCTAC 3 Tm 62 55 C Full length cDNA 4 9 kb gk BioMag mRNA Polysciences NucleoTrape mRNA MACHEREY NAGEL FastTracke 2 0 mRNA Isolation Kit Life Technologies Absolutely mRNA Purification Kit Agilent Technologies S f amp FHU C poly A RNA
23. E RACE PCR RACE PCR PCR RACE C RACE ds cDNA cDNA cDNA RACE PCR RACE PCR 5 10 2 PCR V E RACE cDNA RACE PCR 2 3 8 10 4 RACE
24. EDTA pH 8 0 7 80 ul O7 rx M TE pH 8 0 Vortex 8 15 000 rpm 10 9 0 5ml O RES 3 Z7 Ic ell d e 10 7 5 M Ammonium Acetate 29 ul 2M 11 2 5 99 566 272 5 ul 12 20 C 1 13 15 000 rpm 4C 20 14 15 20C 75 400 ul 16 15 000 rpm 4C 5 17 18 5 10 19 10 ul O TE pH 8 0 20 1 ul ds cDNA 0 2 0 4 ux g ml 250 500 ul TE pH 8 0 cDNA cg
25. Strand cDNA Second Strand cDNA RNaseH coli DNA Polymerase E coli DNA Ligase RNA DNA RNA Second Strand cDNA 1 2 RACE Double Stranded ds cDNA T4 DNA Polymerase ds cDNA ds cDNA RACE 1 5X Second Strand Buffer ANTP Mix MAKKA TRDI o 2 First Strand cDNA First Strand Mix 10 ul TALES ZK 48 4 ul 5X Second Strand Buffer 16 ul dNTP Mix 10 mM 1 6 ul Vortex E coli RNase H 1U l ul E coli DNA Ligase 5U 1 ul E coli DNA Polymerasel 24U 2 yl Total volume 80 ul 3 4 4C 5 16C 1 5 6 1 6 ul O 0 5 M
26. ed Cold Spring Harbor Laboratory Press Cold Spring Harbor New York 16 Kapranov P Cheng J Dike S Nix D A Duttagupta R Willingham A T Stadler P F Hertel J Hackerm ller J Hofacker I L Bell I Cheung E Drenkow J Dumais E Patel S Helt G Ganesh M Ghosh S Piccolboni A Sementchenko V Tammana H amp Gingeras T R 2007 RNA maps reveal new RNA classes and a possible function for pervasive transcription Science 316 1484 1488 17 Mercer T R Gerhardt D J Dinger M E Crawford J Trapnell C Jeddeloh J A Mattick J S amp Rinn J L 2012 Targeted RNA sequencing reveals the deep complexity of the human transcriptome Nature Biotechnol 30 99 104 18 Hirano M amp Noda T 2004 Genomic organization of the mouse Msh4 gene producing bicistronic chimeric and antisense mRNA Gene 342 165 177 19 Hirano M 2008 Natural Grp78 antisense RNA induces apoptosis by creating a chimeric mRNA with Msh4 pre mRNA In A G Hernandes Ed Antisense Elements Genetics Research Focus pp 27 69 Nova Science Publishers Inc New York 20 Sikela J M amp Auffray C 1993 Finding new genes faster than ever Nature Genet 3 189 191 21 Velculescu V E Zhang L Vogelstein B amp Kinzler K W 1995 Serial analysis of gene expression Science 270 484 487 22 Liang P amp Pardee A 1992 Differential display of eukaryotic messenger RNA by means of the polymerase cha
27. in reaction Science 257 967 970 23 Yates L A Norbury C J amp Gilbert R J C 2013 Long and short of microRNA Cell 153 516 519 24 Numata K Kanai A Saito R Kondo S Adachi J Wilming L G Hume D A Hayashizaki Y amp Tomita M 2003 Identification of putative noncoding RNAs among the RIKEN mouse full length cDNA collection Genome Res 13 1301 1306 25 Nam J W amp Bartel DP 2012 Long noncoding RNAs in C elegans Genome Res 22 2529 2540 11 accra expRACE KIT Email contact elplain com Email tech elplain com 4 304 350 U S patent No 7 504 240
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