• 婎杮揑側拲堄
• 慜擔傑偱偺弨旛
  • 婍嬶偺柵嬠
  • 帋栻偺嶌惉
    • A. 幒壏曐懚
    • B. 椻搥曐懚
    • C. 椻憼曐懚
  • 泱偺屌掕
• 堦擔栚屵慜帋栻嶌傝
• 堦擔栚屵屻
  • 慜張棟
  • ProK treatment
  • Hybridization
• 擇擔栚屵慜丂帋栻弨旛
• 擇擔栚屵屻丂PROBE 夞廂
• 嶰擔栚

1. RNase 偺墭愼偑柍偄傛偆偵尩廳拲堄
2. 婍嬶偼慡偰柵嬠偐巊偄幪偰梕婍巊梡
3. 憖嶌偼昁偢庤戃拝梡丄摿偵僒儞僾儖僠儏乕僽偼慺庤偱怗傜側偄丅
4. 帋栻偺検偲忦審乮壏搙側偳乯傪娫堘偊側偄偙偲

RNase free

• dH2O 1l
• PTw 2l
• TAE 200ml
• 20X SSC 50ml
• Hybridization Buffer
• 10% tween
• 10% chaps
• Proteinase K

Others

丂

• 2 x SSC 500ml
• 0.2 x SSC 300ml

慜擔傑偱偺弨旛

• 婍嬶偺柵嬠
  僈儔僗婍嬶丂姡擬柵嬠
  僾儔僗僠僢僋梕婍乮庡偵巊偄幪偰乯
• 帋栻偺嶌惉
  儔儀儕儞僌
  嶌偭偨帋栻偵偼帋栻柤偲挷惢擔傪彂偄偰偍偔丅
  僗儁乕僗偑偁傟偽丄惂嶌幰柤乮傕偟偔偼僀僯僔儍儖乯傕婰擖
• 泱偺屌掕
• 偦偺懠偺幚尡忦審
  幚尡幒丂Wash 傑偱偼RNase 傪巊梡偟偰偄傞晹壆偼巊傢側偄丅
  庤戃丂丂Wash 傑偱偼僒僋儔儊儞偺庤戃拝梡丅
  壏搙丂丂Hybridization, Wash 偺壏搙偼尩庣丄帋栻偼偁傜偐偠傔強掕偺壏搙偵偟偰偍偔

1. DEPC DW(DEPC H2O)
   2L 偔傜偄嶌偭偰偍偔
   DW
   DEPC 0.1% (1ml/l)
   Stir over night
   autoclaved
   
2. 10亊 PBS for 1l
   

   NaCl	Sodium Chloride 墫壔僫僩儕僂儉	58.44	1369mM	80g
   KCl	Potassium Chloride 墫壔僇儕僂儉	74.55	26.8mM	2g
   Na2HPO4	Sodium Phosphate, Dibasic, Anhydrous 儕儞巁堦悈慺僫僩儕僂儉乮柍悈乯	141.96	81.0mM	11.5g
   KH2PO4	Potassium Phosphate, Monobasic 儕儞巁擇悈慺僇儕僂儉乮柍悈乯	136.09	14.7mM	2.0g
   H2O				800ml

   
   adjust the pH to 7.4 then make up to 1l

   autoclave
   

3. PTw for 1l RNase free 2l偔傜偄偮偔偭偰偍偔
   

   10亊 PBS	100ml
   10% Tween 20	10ml(0.1%)

   autoclave after DEPC treatment
   
   
   
4. PBT (BSA-) Final RNase free MAB Buffer 傪巊偆偲偒偼偄傜側偄
   

   10亊 PBS	1l
   Triton(10%)	10ml (0.1%)
   BSA	2g (2mg/ml)巊梡捈慜偵壛偊傞

   
   
   
5. MAB(Maleic acid buffer) antibody wash梡偺Buffer
   

   Maleic acid	116.1	100mM	11.61g
   NaCl	58.44	150mM	8.77g
   H2O			make up to 1l

   
   pH to 7.5 with 10N NaOH (10~22ml)
   
   
   
6. 20X SSC for 1l RNase free
   

   NaCl	Sodium chloride 墫壔僫僩儕僂儉	58.44	3M	175.3g
   NaOCOCH2C(OH) (COONa)CH2COONa �2H2O	Sodium citrate 僋僄儞巁俁僫僩儕僂儉乮俀悈榓乯	294.10	300mM	88.2g
   H2O				800ml

   
   adjust the pH to 7.0 with 1N HCl
   add 1ml of DEPC adjust volume to 1l stir over night
   Sterilize by autoclaving
   
   
   
   
   
7. MetOH series丂丂100ml each
   100% MetOH l
   75% MetOH/25% DEPC dH2O (3:1)
   50% MetOH/50% DEPC dH2O(1:1)
   25% MetOH/75% PTw (1:3)
   
   
8. MEMFA 10ml *巊梡捈慜偵嶌惉偡傞
   10亊MEM 1ml
   37亾Formalin 1mlDEPC DW 8ml
   
   

Optional 晛捠偼巊傢側偄

1. levamisol 100mM in DW (stock solution)
   250mg in 10ml (0.5mg/ml=2mM)
   
2. AP-reaction buffer BM purple 傪巊偆偲偒偼偄傜側偄
   0.1M Tris-HCL(pH9.5)
   0.1M NaCL
   autoclaved
   +12.5mM MgCl2 (巊梡捈慜偵壛偊傞乯
   

儅僀僋儘僠儏乕僽偵暘拲
1. Denhardt's solution 50亊 2ml偱 for 10times
   Ficoll 5g 1%
   polyvinylpyrrolidone 5g 1%
   BSA(Pentax FractionV) 5g 1%
   DW to 500ml
   Filter through a disposable Nalgene filter.
   Dispense into 25ml aliquots.
   
   Store at -20�C
   乮巗斕昳偱傕壜丄榓岝側偳乯
   
   
   
2. 儂儖儉傾儈僪尨塼丂榓岝扙僀僆儞嵪傒 (尨塼偼搥偭偰偄傞偺偱搾愾偱梟偐偡)
   Make 5~10mls aliquots and store at -20�C
   
   
3. MAB-B stock solution (10% Boehringer-Mannbeim blocking reagent in MAB)
   
   Dissolve in MAB by gently heating in microwave, then autoclave.
   Aliquot into tubes and freeze at -20C
   
   
4. heparin (100mg/ml) 10兪l
   
   
   
5. RNase A 10mg/ml RNase 乮Treatment 傪偡傞偲偒偵偺傒巊偆丅乯
   
   Buy solution, its safer than dissolve the powder.
   RNase A偼偙偺擹搙偺梟塼傪攦偭偨曽偑埨慡
   
   10mM Tris-HCl 15mM NaCl pH7.5 偺僶僢僼傽乕偵RNase A 傪擖傟丄暒摣偟偨100偍搾偱搾愾偟偰梟偐偡丅
   椻傑偡偲偒偵偼丄偍搾偵偮偗偨傑傑帺慠偵椻偊傞丅媫椻偡傞偲捑揵傪惗偠偰偟傑偆丅
   
   
   
   
6. Pro K stock solution: 10mg/ml (or 1mg/ml) Proteinase K
   
   
   
   
7. Sheep serum
   
   Inactivate by heating at 56�C for 30 minutes
   Make 2ml aliquotes and store at -80�C.
   
   56亷 偱30暘張棟偟丄2ml偢偮暘拲偟偰偍偔丅
   
   
8. tRNA
   Torula RNA (Type VI, Sigma R 6625) 200mg 傪20ml偺DW偵偲偐偟偨屻(旕忢偵梟偗偵偔偄乯20ml PCI拪弌丄媦傃丄20ml僋儘儘儂儖儉拪弌傪峴偄丄忋惔傪偦偺傑傑1ml偢偮暘拲偟偰偍偔丅
   擹搙偼傎傏10mg/ml偵側偭偰偄傞偼偢丅

丂
1. 10亾 Tween 20
   Tween20 1ml
   DW丂丂丂丂 9ml
   
   
   
2. 10% CHAPS
   CHAPS 1g
   DEPC DW 9ml
   
   
   
3. 10X MEM stock soln. for 100ml RNase free乮幷岝曐懚乯
   

   MOPS	209.27	1M	20.93g
   EGTA	380.35	20mM	0.76g
   MgSO4�7H2O	246.48	10mM	0.25g
   H2O			make up to 100ml

   
   adjust pH to 7.4 after you disolve MOPS
   should be autoclaved
   
   拲堄丗弴斣偵傛偭偰偼EGTA偼夝偗偵偔偄偺偱崿偤傞帪偼pH 傪偁傢偣偰偐傜
   
   
   
   
4. 0.1M TEA(triethanolamine) pH7乣8 (Better to prepare freshly.) for 100ml
   
   TEA 1.5g (1.33ml at 20亷)
   DW 98ml
   conc. HCl 0.5ml丂乮擹墫巁乯
   仺 Check the pH then treat with DEPC
   
   Prepare 100ml X 2 bottles
   
   
   
5. 4% formaldehyde in PTw
   10% formaline in PTw
   

1. Embryo傪僔僗僥僀儞偱張棟偟偰扙僛儕乕偡傞丅
2. 擷泱丒恄宱泱偼價僥儕儞枌傪庢傝彍偒丄旜夎泱偼0.01% MS222偐僋儘儘儂儖儉偱杻悓偡傞丅
3. 俵俤俵俥俙丂侾.5乣俀倛倰丂俼.俿.偱屌掕丅
   丂丂丂丂丂俵俤俵俥俙丂丂丂 10亊俵俤俵 1ml
   丂丂丂丂丂丂丂丂丂丂丂丂丂丂Formalin 1ml
   丂丂丂丂丂丂丂丂丂丂丂丂丂丂DEPC H2O 8ml 偺妱崌偱崿偤傞丅
   
   扙怓偺昁梫偑偁傟偽埲壓偺僗僥僢僾傪壛偊傞丅
   
   
4. 10% H2O2/MeOH乮H2O2丗MeOH亖1丗2乯丂俁倣倢偱俇hous 寀岝摂壓 乮懡彮摦暔嬌偺怓慺傪巆偟偰傕椙偄応崌 3 hours 乯
5. 100% MeOH -20亷偱曐懚
   

H.S.: hybridization solution for 10ml (for 4~5 samples, 2.2ml for one sample)

formamide	5ml	50%
SSC(20亊)	2.5ml	5亊
heparin (100mg/ml)	10兪l	100兪g/ml
Denhardt's (50亊)	200兪l	1亊
Tween 20 (10%)	100兪l	0.1%
CHAPS (10%)	100兪l	0.1%
tRNA(10mg/ml)	100兪l	100兪g /ml
0.5M EDTA pH8.0	200兪l	5mM
DEPC-dH2O	1.79ml


store at 4亷

1. Pre-treatment 慜張棟

   RNase free conditon
   

   1. 100mls of 100% MetOH 5 min with agitation
      
      
      屌掕偟偨泱傪100%儊僞僲乕儖偵怹偟偨僶僗働僢僩偵堏偟偰5暘備偭偔傝偲怳岷丅
      泱偺堏摦偵偼丄愭傪僇僢僩偟偨僽儖乕僠僢僾偐丄愭傪僇僢僩偟偨僷僗僣乕儖僺儁僢僩傪梡偄傞丅
      
      
   2. 100mls of 75% MetOH/25%DEPC-dH2O 5 min with agitation on a mild mixer
      
      
      75% 儊僞僲乕儖25%DEPC忲棷悈偱5暘備偭偔傝偲怳岷
      
      
      
   3. 100mls of 50% MetOH/50% DEPC-dH2O 5 min with agitation on a mild mixer
      
      
      50% 儊僞僲乕儖50%DEPC忲棷悈偱5暘備偭偔傝偲怳岷
      
      
      
   4. 100mls of 25% MetOH/75% PTw 5 min with agitation on a mild mixer
      
      
      25% 儊僞僲乕儖 75% PTw偱5暘備偭偔傝偲怳岷
      
      
      
   5. 100mls of PTw 5 min X 3
      
      
      
      PTw偱5暘備偭偔傝偲怳岷丂3夞孞傝曉偡丅
      
      
      Pro K in PTw 傪嶌偭偰偍偔丄ProK stock(10mg/ml)傪PTw1ml偁偨傝1兪l壛偊傞丅
      ProK (10兪g/ml) 1 sample 偁偨傝0.5ml
      
      
      
      
2. ProK treatment
   
   
   
   1. Transfer the baskes to 15ml round-bottom polypropylene tubes containing
      0.5ml of 10兪g/ml Proteinase K in PTw.
      
      
      0.5ml 偺10兪g/ml Proteinase K PTw梟塼偺擖偭偨15ml偺娵掙億儕僾儘僺儗儞僠儏乕僽偵僶僗働僢僩傪堏偡丅
      
      
      
   2. Incubate 5~10minutes at room temp. agitating occasionally by gently tapping the side of the test tube rack.
      
      
      偲偒偳偒桪偟偔僞僢僺儞僌偱崿偤側偑傜丄5~10暘幒壏偱僀儞僉儏儀乕僩偡傞丅
      
      乮泱偑傕傠偔側傞偺偱尨挵泱 偼憗傔偵斀墳傪掆巭偡傞乯
      
      
      
   3. Transfer the baskets back to rack sitting in 100mls PTw Just rinse
      
      
      僶僗働僢僩傪PTw偵偮偗偰偁傞儔僢僋偵栠偟偰寉偔愻偆丅
      
      
      
   4. 100mls of 0.1M TEA 5min with agitation
      (distroy endogenous alkaline phosphatase)
      
      
      100mls 偺0.1M TEA偱 5暘備偭偔傝怳岷偟側偑傜愻忩偡傞丅
      乮撪嵼惈偺傾儖僇儕僼僅僗僼傽僞乕僛傪暘夝偡傞乯
      
      
      
   5. 100mls of 0.1M TEA 5min with agitation
      
      
      
      100mls 偺0.1M TEA偱 5暘備偭偔傝怳岷偟側偑傜愻忩偡傞丅
      
      
      
   6. Add 250兪l of acetic anhydride 5min with agitation
      
      
      250兪l偺柍悈恷巁傪壛偊偰 5暘備偭偔傝怳岷偟側偑傜愻忩偡傞丅
      乮傾儈僲巆婎傪傾僙僠儖壔偟丄僾儘乕僽偺旕摿堎揑側寢崌傪杊偖丅乯
      
      
      
   7. Add 250兪l of acetic anhydride 5min with agitation
      
      
      偝傜偵250兪l偺柍悈恷巁傪壛偊偰 5暘備偭偔傝怳岷偟側偑傜愻忩偡傞丅
      
      
      
   8. 100mls of PTw 5min with agitation X 2
      
      
      PTw偱5暘娫怳岷偟側偑傜愻忩偡傞丅2夞孞傝曉偡丅
      
      
      
      
   9. 100mls of 4% formaldehyde in PTw 20min with agitation
      
      
      4% 儂儖儉傾儖僨僸僪PTw梟塼偱20暘娫怳岷偟側偑傜屌掕偡傞丅
      
      
      
   10. 100mls of PTw 5min with agitation X 5
       
       
       PTw偱5暘娫怳岷偟側偑傜愻忩偡傞丅俆夞孞傝曉偡丅
       
       
       
3. Hybridization
   
   1. Transfer the baskets to 15-ml snap-cap polypropylene tubes containing PTw 500兪l+H.S 125兪l, then agitate 5min
      
      PTw500兪l+H.S 125兪l偺擖偭偨15ml偺娵掙億儕僾儘僺儗儞僠儏乕僽偵僶僗働僢僩傪堏偟丄5暘怳岷偡傞丅
      
      
      
   2. 500兪l H.S. 10min at 60亷 with agitation in hybridization oven.
      
      500兪l 偺僴僀僽儕僟僀僛乕僔儑儞塼(H.S.) 拞偱60亷10暘娫怳岷偡傞丅
      
      
      
   3. 500兪l H.S. 6 hours at 60亷 with agitation in hybridization oven.
      (pre-hybridization).
      
      
      500兪l 偺僴僀僽儕僟僀僛乕僔儑儞塼(H.S.) 拞偱60亷6帪娫怳岷偟側偑傜僾儗僴僀僽儕僟僀僛乕僔儑儞傪偍偙側偆丅
      
      
      <捈慜偵Probe in H.S 傪嶌傞丅
      85亷丄2乣3min偱denature 仺on ice 5min>
      
      
      
      
   4. Probe (0.3~0.5兪g) in 500兪l H.S.
      
      
      僾儘乕僽擖傝偺僴僀僽儕僟僀僛乕僔儑儞塼偵堏偡丅
      
      
      
   5. Agitate overnight (12~16 hours) at 60亷 in hybridization oven.
      
      怳岷偟側偑傜堦斢僴僀僽儕僟僀僛乕僔儑儞傪偍偙側偆丅
      
      
      
      

• 2 X SSC 300~500 ml 60�C
  
• O.2 X SSC 200~300ml 60�C
  
• MAB-B
  

弨旛丗

2X SSC 300ml 傪60亷偵壏傔偰偍偔丅 乮僂僅乕僞乕僶僗偑椙偄乯

0.2X SSC 200ml傪60亷偵壏傔偰偍偔丅乮僂僅乕僞乕僶僗偑椙偄乯

• 4 愻偄偲僽儘僢僉儞僌
  
  
  1. 僾儘乕僽偺擖偭偨H.S.傪夞廂偡傞丅
     
     
  2. 500兪l H.S. 10 min at 60亷 with aggitation in hybridization oven.
     
     
     
  3. 100mls of 2亊SSC丂20min at 60亷丂亊3
     
     
     僶僗働僢僩傪儔僢僋偵栠偟偰60亷偺100ml 2亊SSC偱20暘愻偆丅3夞孞傝曉偡丅僂僅乕僞乕僶僗傪巊偆丅傑偨丄壏搙掅壓傪旔偗傞偨傔偵億儕梕婍偺奧傪偟偰偍偔丅
     
     
     
  4. 100mls of preheated 0.2X SSC and agitate for 30 minutes at 60亷
     
     
     
     
  5. 100mls of preheated 0.2X SSC and agitate for 30 minutes at 60亷
     
     
     
     
  6. 100mls of MAB (maleic acid buffer). Agitate for 15minutes at room temperature.
     
     
     100mls 偺儅儗僀儞巁僶僢僼傽乕偱幒壏偱怳岷偟側偑傜15暘娫愻忩偡傞丅
     
     
     
     
  7. 100mls of MAB (maleic acid buffer). Agitate for 15minutes at room temperature.
     
     
     MAB-B傪梡堄偟偰偍偔乮僔儍乕儗梡偼10~15ml掱搙乯丅仺MAB-B stock solution 傪MAB偱5攞婓庍
     
     
     
     
  8. Transfer the embryos to glass vials containing O.5ml of MAB-B (MAB containing 2% BM Blocking reagent).
     
     Agitate 1 hour at room temperature
     1帪娫幒壏偱備偭偔傝偲怳岷偡傞丅
     
     
     <泱傪僶僀傾儖偵堏偡偲偒偼丄愭偢MAB-B傪僈儔僗僔儍乕儗偵枮偨偟偰偍偄偰丄偦偙偵僶僗働僢僩偐傜泱傪僔儍乕儗偵堏偡丅>
     
     亙偙偺娫偵MAB-B containing 20% heat-inactivated sheep serum 傪梡堄偟偰偍偔丅MAB-B stock : serum : MAB =1:1:3 1 ml for 1 sample 亜
     

     丂

  9. Aspirate off the solution and replace with 0.5mls MAB-B containing 20% heat-inactivated sheep serum.
     In order to prevent destruction, keep the embryos submerged.
     
     Incubate for 1hour at room temperature with agitation.
     
     
     <姰慡偵塼傪彍偔昁梫偼側偄丅泱偼夡傟傗偡偔側偭偰偄傞偺偱丄塼偵怹偭偰偄傞忬懺傑偱塼傪巆偟偰偍偄偰丄塼傪壛偊傞丅塼偼丄僶僀傾儖偺暻柺傪偮偨偆傛偆偵偟偰拲偖丅埲壓摨條偵憖嶌丅>
     
     
     幒壏偱1帪娫怳岷偟側偑傜張棟偡傞丅
     
     
     
  10. Remove solution by aspiration and add 0.5ml MAB-B/20% sheep serum containing 1/2000 dilution of alkaline phosphatase-conjugated anit-DIG antibody.
      
      Incubate overnight with agitation at 4亷 (in a cold room).
      
      
      塼傪彍偒丄1/2000偺擹搙偱乽傾儖僇儕僼僅僗僼傽僞乕僛傪寢崌偝偣偨峈DIG峈懱乿傪娷傫偩 MAB-B/20% sheep serum 0.5ml 傪壛偊傞丅
      
      
      4亷 偱堦斢斀墳偝偣傞丅

  
  
  

• 5 Antibody wash
  
  
  1. Aspirate off the antibody solution and rinse embryos in MAB.
     
     峈懱塼傪偺偧偒丄1ml 偺MAB偱5暘掱搙愻偆丅乮俀夞傎偳愻偭偨曽偑椙偄応崌偑偁傞丅乯
     
     
     
  2. Fill vial with MAB and agitate slowly for 1hour at room temperature. X5
     
     MAB 偱僶僀傾儖傪枮偨偟丄1帪娫備偭偔傝偲怳岷偟側偑傜愻偆丅
     偙傟傪慡晹偱5夞孞傝曉偡丅
     
     
     
• 6 Coloring reaction with BM purple
  
  
  1. Remove the MAB and add 0.5 ml BM purple cover with alminum foil and incubate for 5min.
     僺儁僢僩儅儞傪巊偭偰MAB 傪彍偒丄0.5ml 偺BM purple乮傾儖僇儕僼僅僗僼傽僞乕僛偺婎幙乯傪壛偊丄傾儖儈儂僀儖偱幷岝偟偰備偭偔傝偲5暘偍偔丅
     
     
  2. Remove BM purple and add 0.5 ml of new BM purple.
     
     BM purple 傪怴偟偄傕偺偵姺偊傞丅
     
     
  3. Cover the vial with alminum foil to cut lights
     
     傾儖儈儂僀儖偱僇僶乕傪偟偰幷岝偟丄敪怓偝偣傞丅
     
     
  4. Check the embryos for staining repeatedly and stop the reaction when the staining condition is apropriate with 1ml of MEMFA or 4% formaldehyde in PBS.
     
     偲偒偳偒敪怓偺嬶崌傪妋偐傔丄揔摉側帪娫偱MEMFA 偐 4% formaldehyde PBS傪壛偊偰敪怓傪巭傔傞丅偩偄偨偄4帪娫傪栚埨偲偟偰敪怓偺嬶崌傪妋偐傔丄僶僢僋偑崅偔側偗傟偽20帪娫掱搙敪怓偝偣偰傕峔傢側偄丅偟偐偟丄probe偵傛偭偰偼1帪娫埲撪偵敪怓偑巒傑傞傕偺傕偁傞偺偱拲堄偡傞丅
     
     
  5. Store in Methanol.
     
     儊僞僲乕儖拞偱曐懚偡傞丅
     
     

  
  

• 6乕A, 敪怓 (NBT,BCIP巊梡乯
  

  AP-reaction Buffer / 1mM levamizol 2ml 5min 亊 2
  NBT4.5兪l/ml +BCIP 3.5 兪l /ml in AP-reaction Buffer 1ml
  5ml~1day
  actin probe 偱2乣3hr
  N-CAM丒Sek 偱3乣4hr
  goosecoid 偱4乣5h
  MEMFA 2ml 2~3hr
  MetOH
  
  

• 6乕B, 敪怓 (寀岝婎幙巊梡乯
  
  Buffer ( 100mM tris-HCl / 100mM NaCl / 10mM MgCl2, pH8.0 仺僼傿儖僞乕偱鄅夁 )
  3ml 30min 亊 2
  HNPP-Fast Red TR斀墳塼偱敪怓 3ml 30min
  
  HNPP (10mg / ml in DMF) 30兪l
  Fast Red TR (25mg / ml in DW) 30兪l 仺僼傿儖僞乕偱鄅夁
  Buffer 3ml
  丂丂丂
  丂丂丂 Buffer偱Wash 3ml 5min
  丂丂丂 敪怓 3ml丂丂丂丂 30min
  Buffer偱Wash 3ml 5min亊 2
  
  

*敪怓偼埫強偱偍偙側偆

1. Linearize 20microgram plasmid DNA with an enzyme which cuts 3' to the transcript of interest.
2. PCI extract the DNA three times.
3. Back-extract the organic layers with DEPC-treated H2O and combine.
4. Chloroform extract once.
5. Isopropyl alchoohol precipitae with NH4OAc, pellet the DNA by centrifuge and discard supernatant.
6. Wash with RNase free 70% ethanol and dry.
   
   拲堄丗惂尷峺慺偵偼丄5'撍弌枛抂傪惗偠傞峺慺傪梡偄傞丅3'撍弌枛抂偺応崌RNA polymerase 偑DNA 偐傜棧傟偵偔偔側傝丄揮幨岠棪偑旕忢偵埆偔側傞丅

In vitro transcription of probes

1. Resuspend DNA in 10兪l of DEPC-H2O
2. Check DNA concentration
3. Mix the following (total volume 20兪l)
   2~4兪g of DNA
   4兪l 5X in vitro stanscription buffer
   2兪l 10X DIG-rUTP/rNTP
   1兪l 0.1M DTT
   1兪l RNA Guard
   1兪l RNA polymerase
   DEPC-H2O to20兪l
4. Incubate at 37�C for 2 hours.
5. Add 1 兪l RNase-free DNAse I and incubate 37�C for 30 minutes.
6. Add
   15兪l NH4OAc
   115兪l DEPC H2O
   150兪l Isopropyl alcohol
7. Mix and put in -20�C for 1hour.
8. Spin in microfuge at 4�C for 10minutes
9. Discard sup. and wash with RNase free 70% ethanol
10. Dry briefly the pellet and resuspend in 10~20兪l of DEPC-H2O
    
    拲丗ROCHE偺DIG RNA Labeling kit 傪梡偄偰傕傛偄偑丄DNAse I 張棟傪峴偭偨屻昁偢恷巁傾儞儌僯僂儉偱捑揵偝偣傞偐丄僎儖傠夁偺僗僺儞僇儔儉傪巊偭偰枹斀墳偺NTPs傪彍偔丅彍偐側偄偲丄旕摿堎揑側敪尰偑弌偰偟傑偆丅

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