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DNA Footprinting DNA Footprinting

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Background on DNA Footprinting Pozadina na DNA Footprinting

DNase footprinting is a molecular biology method that enables the detection of DNA-protein interactions by exploiting the property that a protein interacting with DNA will protect the DNA at that interaction site from restriction digestion or DNAase enzymatic cleavage. DNase footprinting je metoda molekularne biologije koje omogućuje detekcija DNA-protein interakcije od strane iskorištavanja imovine da protein interakcije s DNA će štite DNA i da interakcija site iz ograničenje probavu ili DNAase enzimska cleavage. A DNA restriction fragment which contains the specific binding site is labeled at one end, usually with 32P and used for footprinting. A ograničenje fragment DNA koji sadrži posebna obvezujuća site je etiketom na jednom kraju, obično s 32P i koristi se za footprinting.

Protein and DNA are allowed to hybridize and bind together. Proteina i DNA je dozvoljeno hybridize i svezati zajedno.

DNA footprinting utilizes the enzyme DNase I or d eoxyribo n ucleic acid nucle ase I in order to digest or cut away free radiolabeled (or labelled) DNA leaving the protein bound DNA protected. DNA footprinting koristi se enzim DNase I ili d eoxyribo n ucleic kiselina nucle ase sam kako bi digest ili cut daleko besplatno radiolabeled (ili označena) DNA ostavljajući protein dužni DNA zaštićena. The DNA restriction fragments are treated lightly with DNase I, which makes single-strand breaks (nicks) in the DNA. DNA je ograničenje fragmenti se tretiraju lako s DNase I, što čini jednim break-obala (nicks) u DNA. A small amount of enzyme is used such that there is an average of less than 1 nick/strand. Malu količinu enzim se koristi tako da je prosječno manje od 1 nick / obala. The reaction is then stopped, the DNA is denatured, and the mixture is run on a denaturing polyacrylamide sequencing gel. Reakcija je onda prestali, DNA je denaturirani, a mješavina je izvoditi na denaturiranje polyacrylamide gel redoslijed. These fragments separated by gel electrophoresis are exposed to detect the protected DNA area by analyzing the banding cleavage pattern on the gel. Ovi fragmenti separated by gel-elektroforeza su izložena otkriju zaštićenog područja DNA analizom je banding cleavage pattern na gel.

The cleavage pattern of the DNA in the absence of a DNA binding protein, typically referred to as free DNA, is compared to the cleavage pattern of DNA in the presence of a DNA binding protein. The cleavage pattern of DNA u odsustvu DNA binding protein, obično zvane slobodna DNA, je u odnosu na cleavage pattern of DNA u prisutnosti DNA binding protein. If the protein binds DNA, the binding site is protected from enzymatic cleavage. Ako se protein veže DNA, binding site je zaštićena od enzimska cleavage. This protection will result in a clear area on the gel which is referred to as the "footprint". Ova zaštita rezultirat će jasno područja na kojima je gel koji se navode kao "noge".

By varying the concentration of the DNA-binding protein, the binding affinity of the protein can be estimated according to the minimum concentration of protein at which a footprint is observed. By različitih koncentracija DNA-binding protein, obvezujući afinitet za protein može se procjenjuje u skladu s minimalnim koncentracija proteina na jedan trag koji je promatrao.

DNAse Footprinting Buffer Recipes DNAse Footprinting Buffer Recepti

DNase Footprinting Binding Buffer Recipe DNase Footprinting Binding Buffer Recipe

2X w/o KCl / For 10mls: 2X w / o KCl / Za 10mls:

amount: [final] iznos: [final]
Tris-HCl pH7.6: 500 ul of 1M: 50 mM Tris HCL-pH7.6: 500 ul. od 1M: 50 mm
MgCl2: 125ul of 1M: 12.5mM MgCl2: 125ul od 1M: 12.5mM
EDTA: 2 ul of 0.5M: 1mM EDTA: ul. 2, 0.5M: 1mM
Glycerol: 2 mls: 20% Glicerola: 2 mls: 20%
DTT : 10 ul of 1M: 1 mM DTT: ul. 10, 1M: 1 mm

Binding Buffer (Gel Shift buffer) Obvezujuća Međuspremnik (buffer Gel Shift)

5X w/o KCL 5x w / o KCL
[final]: ingredient [final]: sastojak
20%: gylerol 20%: gylerol
5mM: MgCl2 5mM: MgCl2
2.5mM: EDTA 2.5mM: EDTA
2.5mM: DTT 2.5mM: DTT
250mM: NaCl 250mM: NaCl
50mM: Tris-HCL, pH 7.5 50mm:-Tris HCL, pH 7,5
0.25mg/ml: poly (dI-dC) poly (dI-dC) 0.25mg/ml: poly (di-DC) poly (di-DC)

Ca/Mg Buffer Ca / Mg Buffer

For 10mls Za 10mls
CaCl2: 50 ul of 1M: 5 mM CaCl2: ul. 50, 1M: 5 mm
MgCl2: 100 ul of 1M: 10 mM MgCl2: 100 ul. od 1M: 10 mm

Loading Solution Loading Solution
0.1 M: NaOH:formamid (1:2 v/v) 0,1 M: NaOH: formamid (1:2 v / v)
0.1%: xylene cyanol 0,1%: xylene cyanol
0.1%: bromophenol blue 0,1%: bromophenol plava

Stop Buffer Stop Buffer
10mls
NaCl: 400 ul of 5M: 200 mM NaCl: 400 ul. od 5m: 200 mm
EDTA: 600 ul of 0.5M: 30 mM EDTA: ul. 600, 0.5M: 30 mm
SDS: 1 ml 10%: 1% SDS-a: 1 ml 10%: 1%

TEBe (Tris-EDTA-Beta mercap.) TEBe (Tris-EDTA-Beta mercap.)
Tris-HCl, pH 7.6: 500 ul of 1 M: 50 mM HCL-Tris, pH 7,6: 500 ul. od 1 M: 50 mm
EDTA: 2ul of 0.5 M: 1 mM EDTA: 2ul od 0,5 M: 1 mm
Beta-mercaptoethanol: 10 ul : 15mM Beta-mercaptoethanol: ul. 10: 15mM

10 x K buffer 10 x K tampon
for 1 ml za 1 ml
Tris-HCl pH 7.5: 100 ul of 1 M: 100 mM Tris HCL-pH 7,5: 100 ul. od 1 M: 100 mm
MgCl2: 100 ul of 1 M: 100mM MgCl2: 100 ul. od 1 M: 100mm
DTT: 50 ul of 1M: 50 mM DTT: ul. 50, 1M: 50 mm
dH2O: 750 ul dH2O: 750 ul.

DNAse Footprinting Protocol DNAse Footprinting Protocol

The DNA used usually contains the binding site of your protein of interest. The DNA koristi obično sadrži binding site vaše protein od interesa. The DNA is purified, then digested with restriction fragments to be of an appopriate size. DNA je pročišćena, zatim ograničenje sa digested fragmenata da se od jedne appopriate veličina. The DNA fragment is labeled on one end (binding site > 25 bp from end). DNA fragment je s oznakom na jednom kraju (binding site> 25 bp od kraja).

One strand labeling can be accomplised by: Jedan nasukavanje označavanje može biti accomplised / la:

1. isolating the fragment with restriction enzyme(s) containing 5' overhang, labeling with Klenow and the appropriate hot nucleotide. isolating je fragment s ograničenjem enzim (e) sadrži 5 'nadstrešnica, označavanje i Klenow s odgovarajućim hot nukleotidne. Then digest with an enzyme that removes one end. Tada digest s enzim koji uklanja jednoga kraja.
2. isolating a fragment digested with a restriction enzyme that creates 5' and 3' end, labeling with Klenow will only label the 5' overhang. isolating fragment digested s ograničenjem enzim koji stvara 5 'i 3' kraju, označavanje s Klenow će samo natpis na 5 'privjesak.
3. As in "a" but with any enzyme and using polynucleotide kinase to label 3' end (kinase) and digesting off one of the ends with a second (third) restriction enzyme. Kao u "" ali s bilo koje enzim i koristeći polynucleotide kinaza na oznaku 3 'kraju (kinaza) i digesting off jedan od završava s drugi (treći) ograničenje enzim.
   (REMINDER: if labeling with Klenow, at the end of reaction add an excess of cold nucleotide of the same nucleotide that is labeled ( ie if you use dCTP32, add dCTP at the end) to make sure all ends are filled in equally. (PODSJETNIK: ako označavanje s Klenow, na kraju dodati reakcija višak hladno nukleotidne od iste nukleotidne da je etiketom (tj. ako koristite dCTP32, dCTP dodati na kraju) kako bi bili sigurni da sve završava u jednako su popunjena.

For Klenow fragments, 0.3ug bring up to 100 ul in TE, heat kill Klenow at 68 °C for 10 minutes. Za Klenow fragmenti, 0.3ug dovesti do ul. 100 u TE, topline ubiti Klenow na 68 ° C za 10 minuta.

LABELING with one 5' overhang Označavanje s jednim 5 'privjesak

Sample RXN : 15 ng is need for each reaction. Uzorak RXN: od 15 je potrebno za svaku reakciju. If making probe for many reactions JUST increase amount of DNA up to 300 ngs. Ako je stvaranje sonda za mnoge reakcije PRAVEDAN povećati količinu DNA do 300 ngs.
15ng: DNA fragment 15ng: DNA fragment
2 ul: 10x K buffer 2 ul.: 10x K tampon
5 ul: 32P dCTP 3.33 uM 10 uCi/ul 5 ul.: 32P dCTP 3,33 um 10 Uči / ul
2 ul: 2mM @ dA, dG dTTP 2 ul.: 2mM @ DA, dG dTTP
1 ul: Klenow Ul. 1: Klenow
20 ul Final volume, 37°C 30 min. 20 ul. Final volume, 37 ° C 30 min.
--add 1 ul 10mM dNTP, 5 min @ 37°C . -- dodati 1 ul. 10mm dNTP, 5 min @ 37 ° C.

--add 80 ul TE. -- dodati 80 ul. TE. 68°C 15 minutes, put on ice. 68 ° C 15 minuta, stavio na led.
--G-50 Spin Column (spin at about 2000g) -- G-50 Spin Stup (spin oko 2000g)
--ADD 1/10 vol 3M Na0Ac, 2.5 volumes EtOH, Ice 30 mins, (Optional if [DNA] is = or > 3 ng/ul you might post G-50 directly) Microfuge 30 minutes. -- Dodaj 1 / 10 vol. 3M Na0Ac, 2,5 svezaka EtOH, Ice 30 mins, (Opcionalno, ako [DNA] = ili> 3 od / ul možda post G-50 direktno) Microfuge 30 minuta.
--wash with 70% -- opere sa 70%
--Dry Bring up in 5ul -- Suhi Dovedite se u 5ul

Binding Reaction For DNase Footprinting Obvezujuća Reaction Za DNase Footprinting

Binding Reaction should have between 10 and 150mM KCl (more salt reduces binding but increases specificity). Obvezujuća Reaction bi trebao imati između 10 i 150mm KCl (više soli smanjuje obvezujući, ali se povećava specifičnost). The typical concentration is 50mM. Tipična koncentracija je 50mm.

Protein DNA Binding: Protein-DNA Binding:
25 ul of 2X Binding Buffer w/o KCl Ul. 25, 2X Binding Buffer w / o KCl
5ul of 3 ng/ul unilabeled DNA 5ul od 3 od / ul unilabeled DNA
X ul of KCl to equal 10-150 mM KCl X ul. od KCl na jednaku 10-150 mm KCl
dH20 to equal 50 ul minus volume for binding protien dH20 na jednaku 50 ul. minus obujam obvezujuća za protien
1-3 Footprinting Units of DNA binding protein (or 10 to 160 ug of Crude nuclear extract) 1-3 Footprinting jedinica DNA binding protein (ili 10 do 160 ug sirove nuklearna izvuci)

--mix gently, ice 10 minutes. -- MIX nježno, ice 10 minuta.

KEEP TIMING IN MIND, KEEP vremena na umu,
--ADD 50 ul of RT Ca/Mg solution, 18°C for 1 minute. -- Dodaj ul. 50, RT Ca / Mg rješenje, 18 ° C za 1 minutu.
--ADD 3 ul dilute RQ1 DNase (0.05 u/ul diluted in Tris) INCUBATE 1 minute. -- Dodaj ul. 3 razrijediti RQ1 DNase (0,05 u / ul razrijeđen u Tris) izleći piliće 1 minute.
--STOP addition of 90 with 37°C STOP solutions, -- STOP dodatak 90 s 37 ° C STOP rješenja

--you should Phenol: CIA, and CIA extract, and Ethanol precipitate. -- trebali Phenol: CIA, CIA i izvuci, i Etanol precipitate.
--RESUSPEND in 4ul of Loading Solution. -- RESUSPEND u 4ul od Loading Solution.
--Run on 5% standard Urea-DNA sequencing gel (consider wedge gel) with appopriate loading lanes such as DNA ladder, uncut DNA, and controls. -- Run na standardnoj 5% Karbamid-sekvenciranje DNA gel (razmislite o klin gel) sa appopriate učitava stazama, kao što su DNA ladder, neobrezani DNA, i kontrole.

Analyze footprinting pattern. Analizirajte footprinting pattern.

Troubleshooting and Problems? Otklanjanje poteškoća i Problemi? See the DNAse Footprinting Forum! Pogledajte DNAse Footprinting Forum!

For problems and troubleshooting DNAase footprinting, post a new thread to discuss it in the DNA Footprinting Forum ! Za probleme i rješavanje problema DNAase footprinting, novi post nit se raspravljati u DNA Footprinting Forum!

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