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Rapid, nonradioactive differential display using Tth polymerase
Tws
TECHNICAL
X to : ~1of11~ IYXpnxluct
2 ..~&I ~imntn~ twfk~ ( 1 I-H’.) gel npp3r,ltus of the DNA sequencer. .i 5I:x tvith 1 pl of loading buffer consisting of 9G% fonnilmide and 10% 131ucdextnn (Pharmacia ). .d ~cn:~ruw for -1min :u 9ST. rhcn chill quickly on ice tid samples on tlw 8% denaturing ge!, L’w 100. 150 and EOOirp tlurwc*wcnt wc mnrken tPhorm:~cra Biotechl. 5 Kun the D.SA scqucncer 0 I?IC Iluorw~nr gel cl&~ ohtained hy the AL.F DNA .quencer Ii are collected and analyzed automatically by the Fnwment ~anagc~ progt~m (Phannacia Hiotech). E..:h fluowscent peak is quantitated in rerms of size tbp,, peztk height and peak area. In the ~3.w of twcrJmplil?ed pnxluc~s which would give unrelizhle area value. an aliquot of the ICR products should he diluted In dlsttlllccl wstcr and reanalyzed
R’c thank Alan J. Kinniburgh. Roswell Park Cancer Institute. Buffalo. NY, USA for his critical reading of this manuscript and ~;en ~+s+x Pharmrlcia Biotwh Japan. Tokyo, for helpful suggestions.
Sam&a 1 1
3
5
L---_-----TIC; 0UOW.K 1996 VW.. 12 NO. 10
396
1”
I
1
Sample 2 n 3
5
,
2”
M
I ~loao
/
d&500 do&
/
- 200 -100
!
------
Putignani. Furlo
Han+.\!.
.\luellcr
(:oluz/l
;trr help
\vvirh rhr manuscript.
inexpensive,home-made DNA size Standanl DNA gel electrophoKsis is an cwznthl method in molecular hioloby. Size standards are wsetl IO idemily separared DNA tkzg menus. Diverse types of DNA size standards are available commercially hut all arc espensive. Fifty microgmms of the popular @X174 digested with HaelI cos& around USSlOO. Dkscouns for larger quantitw rx~ly exceed 25%. In Europe prices are mon :han 1.5 times as high. Plasmids present in the laboratory cm he digested with rt-striction cnqmes .mtl compaxi to cst3hlished size rtand;irds hJ gel eletlttoph&esis. Plasmids with s&taht pd’-terns czn be preparatively digested and used as DNA size standarcis. Among the plasmids in our laboratory a eukaryotic expression vector containing the P-galactosidase gene @CMVp) ws bP identikd as the most (Fig. 11. _I353 2176One liter of backrid cu!tun2 can yield mg or 970-1078 more of pfasmid DNA, Preparing plasmid DNA -872 75Q1033takes a couple of hour?;. Using the polyethylene glycol method the cost of tnatetial Ls negligiblet. To digest 1 mg of plasmid DNA with a common I5445 -603 653bc' -587 enzyme like H~wIII. 1000 u&s aw required 3t 3 price of USSIOD. To huy mg of ~~~mrnercial -434 305 -310 394DNA star&xl woul~l wu aw!.G ti times as much. 23% '94ij-g -194 298-267 Custs of home-made and commercial size standards per gel lane are USSO. and 2. rqxctiwly.
appropriate
about
bp
bP
I
-184
154-
f&FERESCE 1
I
!
-124
Sambrook. J , Fritxh. E.F. and Manintts. T. ( 1989) Mo~ecdar Cloninfg. A Lukorcrto~ ~Wwtnl, Cold Spring Harhor Labontory Press Ftcuw 1. pCMV!3 was digested n11h HrrcllI and m~lyzcd on I% agarox gels. @X174. digested Rirh M&II. and DIVA molecular weight markers (MYVMj V and Vl @xhringer hfannhrim. Germany) xvere included for compxison. Sizes of bands (hp) are indkttrc!. Laws I snd 4, pC.Clv@digesrrd wth ffrieiI1. lane 2. @X174 rligr9ed n lth Ilf~411: lane 3. MM1 VT; lane 5. wxr V.
TIC
OC.TOWR 1YY6 Vat.
397
I2 No. IO
,
TECHNICAL
X to : ~1of11~ IYXpnxluct
2 ..~&I ~imntn~ twfk~ ( 1 I-H’.) gel npp3r,ltus of the DNA sequencer. .i 5I:x tvith 1 pl of loading buffer consisting of 9G% fonnilmide and 10% 131ucdextnn (Pharmacia ). .d ~cn:~ruw for -1min :u 9ST. rhcn chill quickly on ice tid samples on tlw 8% denaturing ge!, L’w 100. 150 and EOOirp tlurwc*wcnt wc mnrken tPhorm:~cra Biotechl. 5 Kun the D.SA scqucncer 0 I?IC Iluorw~nr gel cl&~ ohtained hy the AL.F DNA .quencer Ii are collected and analyzed automatically by the Fnwment ~anagc~ progt~m (Phannacia Hiotech). E..:h fluowscent peak is quantitated in rerms of size tbp,, peztk height and peak area. In the ~3.w of twcrJmplil?ed pnxluc~s which would give unrelizhle area value. an aliquot of the ICR products should he diluted In dlsttlllccl wstcr and reanalyzed
R’c thank Alan J. Kinniburgh. Roswell Park Cancer Institute. Buffalo. NY, USA for his critical reading of this manuscript and ~;en ~+s+x Pharmrlcia Biotwh Japan. Tokyo, for helpful suggestions.
Sam&a 1 1
3
5
L---_-----TIC; 0UOW.K 1996 VW.. 12 NO. 10
396
1”
I
1
Sample 2 n 3
5
,
2”
M
I ~loao
/
d&500 do&
/
- 200 -100
!
------
Putignani. Furlo
Han+.\!.
.\luellcr
(:oluz/l
;trr help
\vvirh rhr manuscript.
inexpensive,home-made DNA size Standanl DNA gel electrophoKsis is an cwznthl method in molecular hioloby. Size standards are wsetl IO idemily separared DNA tkzg menus. Diverse types of DNA size standards are available commercially hut all arc espensive. Fifty microgmms of the popular @X174 digested with HaelI cos& around USSlOO. Dkscouns for larger quantitw rx~ly exceed 25%. In Europe prices are mon :han 1.5 times as high. Plasmids present in the laboratory cm he digested with rt-striction cnqmes .mtl compaxi to cst3hlished size rtand;irds hJ gel eletlttoph&esis. Plasmids with s&taht pd’-terns czn be preparatively digested and used as DNA size standarcis. Among the plasmids in our laboratory a eukaryotic expression vector containing the P-galactosidase gene @CMVp) ws bP identikd as the most (Fig. 11. _I353 2176One liter of backrid cu!tun2 can yield mg or 970-1078 more of pfasmid DNA, Preparing plasmid DNA -872 75Q1033takes a couple of hour?;. Using the polyethylene glycol method the cost of tnatetial Ls negligiblet. To digest 1 mg of plasmid DNA with a common I5445 -603 653bc' -587 enzyme like H~wIII. 1000 u&s aw required 3t 3 price of USSIOD. To huy mg of ~~~mrnercial -434 305 -310 394DNA star&xl woul~l wu aw!.G ti times as much. 23% '94ij-g -194 298-267 Custs of home-made and commercial size standards per gel lane are USSO. and 2. rqxctiwly.
appropriate
about
bp
bP
I
-184
154-
f&FERESCE 1
I
!
-124
Sambrook. J , Fritxh. E.F. and Manintts. T. ( 1989) Mo~ecdar Cloninfg. A Lukorcrto~ ~Wwtnl, Cold Spring Harhor Labontory Press Ftcuw 1. pCMV!3 was digested n11h HrrcllI and m~lyzcd on I% agarox gels. @X174. digested Rirh M&II. and DIVA molecular weight markers (MYVMj V and Vl @xhringer hfannhrim. Germany) xvere included for compxison. Sizes of bands (hp) are indkttrc!. Laws I snd 4, pC.Clv@digesrrd wth ffrieiI1. lane 2. @X174 rligr9ed n lth Ilf~411: lane 3. MM1 VT; lane 5. wxr V.
TIC
OC.TOWR 1YY6 Vat.
397
I2 No. IO
,