Primer3 http://frodowimitedu/cgi-bin/primer3/primer3_wwwcgi Primer5 Oligo665 Primer5 http://wwwbioonnet/dispbbsasp?boardid=25&id=38536 Oligo667 http://wwwbioonnet/dispbbsasp?boardid=25&id=40898 1 PCR 1 18 24 24 2 GC 40 60 GC GC 3 5' G C 4 3',? G? :? G 0~-2,PCR 100%,? G=-6, 40% 5 3' GC 5 3 A T 6 3' 3' 3 G C AT 7 8 Tm 50 DNA Tm 5 Tm (Wallace ):Tm = 4 g + C +2 a + T, 15-20, (Baldino ) 14-70 04mol\L Tm, Tm Tm=815+166*lg[K + ]+041(%[G+C])-(675/n) Tm GGGAA GAGAG Tm Tm Primer5 1 11
5' Tm 10 3' 3 PCR 1µM 3µM, :,,!, ATG TGA ATG TCA 5 50 OD 1OD 2OD 1OD HPLC OPC http://wwwtakaracomcn/product/cs/c_3htm#1 PCR N PCR 3 PCR 1pg-1 1 mol/l DNA 1 5 Mg 2 15mmol/L dntps KCl 200 mol/l 50 mmol/l 1 PCR PCR 2 11
DNA P DNA PCR DNA SDS 001 100ng 1µg DNA 3 10 4 3 10 5, 01 g DNA,10ng DNA,1ng DNA, PCR,, DNA DNA PCR 2 TE 100µM TE ph TE PCR ddh 2 O -20 10µM TE -20 6 15 30 1-20 1 15 30 2 01 05µM http://wwwtakaracomcn/product/fl/i_1htm 3 Taq DNA 3' 5' 3' 5' Taq DNA Merck Easy-A 3 A 3-5 TA Taq A Taq DNA 3' 5' Taq DNA 4 Mg 2 PCR DNA dntp 200µM dntp PCR 15mM PCR 3 5mM, 3 11
5 dntps dntps dntp 200µM 25-50µM 6 KCl 50 mmol/l, 70-100 mmol/l PCR 1, 94 5-10min, DNA, Taq DNA (hot start),, PCR, DNA, DNA 94 1min, DNA, GC, 2 PCR 55 70 Tm 5,, 2 Tm Tm 5 Tm 5 Tm,, ( 60 94 ),, 3 72, Taq DNA 75,, Taq DNA 20-85,Taq DNA 1kb DNA,, (10-30min),, 72 10min http://wwwbioonnet/dispbbsasp?boardid=95&id=47101 4, DNA 25-30, PCR 25-30, Taq DNA,,, DNA 103-105,, 60, 109-1010, :C C 0 (1+P) n C,C 0 DNA, P, n,,, 4 11
PCR 1 PCR TouchDown PCR PCR PCR Tm 5 1 2 1 2 Tm 5 PCR AFLP DNA 2 PCR PCR Taq DNA 72 PCR DNA PCR Taq DNA PCR PCR DNA PCR Taq DNA 3 PCR GC DNA PCR DMSO PCRx Enhancer Solution DNA PCRx Solution Platinum Taq DNA Platinum Pfx DNA Platinum - Taq DNA DMSO 4 PCR 15 20 100 1000 15 20 PCR 5 11
30 40 PCR mrna PCR 5' RACE PCR PCR : 1 DNA : PCR,,,,,,, 2 PCR :, PCR PCR 1 ( ) : ; ; ( )PCR : PCR PCR ( )PCR PCR PCR ( 1013 /ml) PCR PCR PCR ; 48000 ( ) : 2 1 : PCR PCR PCR (100 ) PCR 6 11
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2 dntp PCR 3 dntp ( ) 1 2 PCR 3 4 dntp 5 6 PCR 7 DNA PCR PCR 8 100 PCR Sourthern PCR PCR DNA PCR 1 2 3 4 5 6 7 1 2 01ml 3 5ml PCR 4 8 8 11
PCR 1 1mol/L DNA 2 UV nm 254/300nm 30min UV PCR PCR UV 500bp UV PCR DNA UV UV DNA 0065/ DNA DNA Taq DNA 013/ DNA 500bp DNA 32 105 100bp 6 105 UV 1 DNase I PCR Taq 05U DNase I 30 min Taq PCR DNA 2 4 Msp I Taq I 1h PCR 3 Taq PCR UV 4 g 15kGy 01ng DNA 20 kgy 104 40 kgy PCR PCR g DNA UNG UV 500bp PCR 300bp UNG 1 PCR du dt du PCR UNG UDG N- du TaqDNA UNG du UNG DNA RNA 2 dutp dutp dttp du PCR UNG PCR PCR UNG PCR du PCR 3 du du dt PCR 5 du UNG 1-2kb dutp dttp du du du 3 4 UNG PCR 9 11
du 5 dutp DNA PCR UNG- UNG UNG 1 RNA 2 3 RNA 2 PCR RS-PCR RNA-specific PCR PCR RNA PCR PCR 3 A 2 0 5 2 0 C mrna cdna C cdna cdna PCR B C cdna DNA DNA DNA 10 11
:, B = C or G or T D = A or G or T H = A or C or T K = G or T M = A or C N = A or C or G or T R = A or G S = C or G V = A or C or G W = A or T Y = C or T PCR PCR RT-PCR AP-PCR PCR PCR invitrogen PCR RT-PCR 3 PCR Football20002 Gaoyx 11 11