12 .有关实时定量 pcr
DESCRIPTION
12 .有关实时定量 PCR. Thanks. - PowerPoint PPT PresentationTRANSCRIPT
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1DNARNA
2 Southern blotNorthern blot
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3 PCR
PCRPolymerase chain reactionDNARNAcDNA106ugDNADNA
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DNADNA3'DNADNADNA4dNTPDNA30-40DNA1pg1ug
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PCR
1PCRPCRTaqMg++dNTPC/G
2PCR
3PCRTaqMg++
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4
1RACE PCR
2CGAT1:1
3RTPCR
5PCRPCR
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6TmPCRTouch Down PCRRACE PCR
7CGDNADMSODIMETHYL SULFOXIDEPCR1/104ulDMSO36ulPCR40ul
8MgCl2PCR bufferMgCl2MgCl2
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9PCRTaq10PCR BufferdNTP
1029
11PCRPCRPCRPCRPCR0.5ml
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12PCR
PCR
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4 PCR
PCRPCR
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PCR
PCRPCRTaqPCRSTRATAGENEQuikChange Site-Directed Mutagenesis KitPCR
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DNAPfuTurbo DNA polymerasebpDNAPCRPCRDpn IDNADpn IDNAPCRPCR
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5 DNA
1DNADDPCR
2
3FootprintDNA
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23ddNTP3DNA5DNA3dNTPDNADNA4dNTPddNTPDNADNAddNTPDNA4ddNTPdNTP4ACGTDNA
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1DNA
1 DNA 12 ul3-5ug
6 ul
2M NaOH/1 mM EDTA 2 ul
37 10 min
2 7 ul
3M NaAC 6 ul
100 75 ul
5 min
310 min470110ul10 min45 min
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4 H2O 6 ul
2 ul
Sequence Buffer 2 ul
65 2 min10-30 min
54 G A T C
Dideoxy Termination Mix ddG ddA ddT ddC
2.5 2.5 2.5 2.5 ul
60.1M DTT 1 ul
Labeling Mix1:4 H2O 2 ul
[35S]dATP 1 ul
Sequenase 2 ul
5 min
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7 G A T C
3.5 3.5 3.5 3.5 ul
3710 min
Stop Solution 4 4 4 4 ul
75-803 min
4
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2
1
1005ml Repel-silane1000.4mmSpacer
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26
5TBE 12 ml
28.8 g
48 ml
40 9 ml
10 0.42 ml
60 ml
TEMED 18 ul
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3
DNA
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3
1
1TBE1000ml551500V
2
GATCGATC2.5ul2000V
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4
1
8080-100
2
XX3
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1
2
3
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44022
5GATCGATC4GCGC
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mRNA
6 RNA
7 mRNA
8 Northern blot
9 RTPCR
10
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mRNA
11 cDNA Array Micro Array
cDNA Array cDNA96cDNA
MicroArraycDNA Array
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MicroArrayRNAcDNARNA
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GeneChip139000216-20mRNA2516-20discoveryARRAYRFDD-PCRPCRMicroArraymRNAPCRmRNAArray
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1
2
3RNA
4
5
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1
2
3
4
5
6
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cDNA ArrayCLONTECH LaboratoriesAtlas cDNA Expression ArrayscDNA ArraycDNA96RNAmRNAmRNAcDNAcDNAArray123
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1RNA2Array34Array1200mRNA
ArrayArrayMicroArrayArrayArrayArray
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cDNA Array9616000Array
cDNA ArraySouhtern blotNorthern blotcDNA ArrayCLONTECHAtlas cDNA Expression Arrays
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CLONTECHDNADNAcDNAmRNAGenBank
1ugmRNAcDNAcDNA ArraycDNA
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X
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1cDNA ArraysmRNAmRNAmRNA
2RNAmRNAmRNAmRNAmRNA
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3[-32P]dATP[-33P]dATP[-32P]dATP[-33P]dATP
4DNAExpressHyb
5
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mRNA
12 DDPCR
DDPCR
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RNAH-T11-CH-T11-GH-T11-A11TmRNAAmRNAcDNAcDNAAAGCTTGATTGCCPCRcDNA[33P]dATPPCRPCRmRNA
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1
Hpa IIpBR 3225GC
pBR3221ul
5RT Buffer4ul
0.1 M DTT0.5ul
[32P]dCTP5ul
2mM dA/T/GTP0.5ul
AMV RT1ul
4230min.
2mM dNTP1ul
4210min.
0.5M EDTA1ul
TE108ul
6510min. 45min.
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Sephedex G502000rpm3
100ulpBR 322
cpm1ul3-4105
2DDPCR
1RNAcDNA
0.5mlul
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1Z1 2Z2
1 2 3 4 5 6
9.4 9.4 9.4 9.4 9.4 9.4
5RT Buffer 4 4 4 4 4 4
dNTP250uM 1.6 1.6 1.6 1.6 1.6 1.6
RNA0.1ug/ul 22 2 2 2 2
H-T11-M2ul A C G A C G
655min3710min
MMLV 1 1 1 1 1 1
3750min755min
4DDPCR
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2DDPCRPCR
1PCR
60
10PCR Buffer 12
dNTP25uM 9.6
-[33P]dATP 1.5
Taq 1.2
20.5ml
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1H-AP-1
1-1 1-2 1-3 1-4 1-5 1-6
H-AP-1 2 2 2 2 2 2
H-T11-M2ul A A C C GG
2ulZ1Z2Z1Z2Z1Z2
PCR 14 14 14 14 14 14
18-32
20 20 20 20 20 20
PCR
94 30 s.
40 2 min.
72 30 s.
40
72 5 min.
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3
3.5ul2ul802min.X
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4DNA
XXDNA300-500bp3MMX
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1.5ml100ul302022-5ulPCRPCR40ulDDPCRPCR200ng-1000ngPCR1Northern blot2PCR
GenBankcDNAcDNA
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1RNA
2DNARNAGHMessage-Clean kitRNADNAmRNAmRNA
3DDPCR100bp1-2
4DDPCR
5DDPCRNorthern blot
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13 cDNA
14 cDNA
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15 RACE PCR
RACE PCRRACERapid Amplification of cDNA EndscDNAPCRRACE PCRPCRcDNA
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cDNA53cDNAPCRPCRPCRcDNAcDNANorthern blotcDNARACE PCR
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1PCR
2123
1PCR23PCRPCRPCRcDNAcDNA
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1PCRRACEcDNAcDNAPCRcDNARACE PCR
2Touchdown PCRTouchdown PCRPCRPCR5~10PCR/3~108078767472
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3PCRPCR
4Tm7022mers25~30mersC/G45~60%Tm
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16 Western blot
17 ELISA
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18
19
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20
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21
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GnRH
GnRH
GnRH GnRH
Chart1
20
130
10
240
30
Sheet1
201301024030
Sheet1
Sheet2
Sheet3
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22
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23 Footprint
24
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25
26
27 DNA
28 DNA
29
DNA
30DNA