Team:NU Kazakhstan/Protocols

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Revision as of 17:54, 18 August 2013

NU_Kazakhstan

SELEX procedure

Materials:

Binding buffer: 50mM Tris-HCl, pH 7.5, 25mM NaCL, 5 mM MgCl2, 10mM dithiothreitol (DTT);

Wash buffer is same as binding buffer;

Elution buffer: 0.4 M sodium acetate, 5mM EDTA, and 7M urea, pH 5.5;

MF-Millipore membrane, mixed cellulose esters, hydrophilic, 0.45 μm, 13 mm. white, plain (HAWP filter, 0.45 μm,13 mm diameter, Millipore) – 2 pcs;

Filter holder – 2 pcs

ssDNAs library was dissolved in sterile water, and concentration was 1 μg/ μl;

Target protein

Petri dish – 1 pcs

6% TBE gels 1.0 mm, 10 well

Syringe with a needle – 2 pcs

glycogen (20 mg/ml); ammonium acetate (7.5 M)

Ethanol (100% and 75%)

10*PCR buffer with MgCl2; dNTPs (2.5 mM); DL-F (20 μM); DL-R phophorylated (20 μM); Taq Polymerase; Lambda exonulcease

Equipments:

Thermocycler

Rotator with variable speed (15 rpm) for 1 ml tubes

Freezers (-20 and -80oC)

Centrifuge – 13000rpm

Buffers for SELEX

Binding buffer/Wash buffer:

DTT- Dithiothreitol, Tris-HCl pH 7.5

Tris-HCl (50mM) – 7.88g for 1L

NaCl (25mM) – 1.461g for 1L

MgCl (5mM) – 0.476g for 1L

DTT (10mM) – 1.5425g for 1L

Elution buffer:

Sodium acetate (0.4M) – 32.812g for 1L

EDTA (5mM) – 1.86g for 1L

Urea (7M) pH 5.5 – 420.42g for 1L

Ammonium acetate (7.5M) – 578.7g for 1L/ 28.9g for 50ml

Procedure

1. Pre-wetted

Soak membrane in sterile water for 1min and put membrane in the filter holder;

To eliminate DNAs that bound to the membrane, DNAs (1 μg/μl) were passed three times prior to the selection cycle through pre-wetted nitrocellulose acetate membranes in “Pop-top” filter holders;

2. DNA denature

For the first cycle of selection, 35.5 μl (1μg/ μl) DNAs were added in 114.5 μl binding buffer solution (volume 150 μl);

And then were denatured at 95oC for 10 min;

Then were allowed to cool in thermocycler to approximately 300C (about 1.5 hours);

3. Binding

Denatured DNAs (35.5 μl, 1μg/ μl DNAs + 114.5 μl binding buffer) + 50 μl target protein (87 μg/ml) were incubated at 15 rpm on a variable speed rotor for 1 hour and 45 min at room temperature.

4. Washing

Soak membrane in wash buffer (the same as binding buffer) for appr. 1 min and then put the membrane in the filter holder;

Aspirate the binding reaction mixture (DNAs+target) with a syringe with a needle

Remove the needle and inject the binding reaction mixture into the filter holder with a membrane and then inject 1 ml wash buffer solution (the same as binding buffer) and let wash buffer pass through the membrane.

5. Elution

Heat 200ul elution buffer(0.4 M sodium acetate, 5 mM EDTA, 7M urea, pH 5.5) to 70-800C

Inject 200 μl of elution buffer into the filter holder to elute DNA that was retained in the filter into a fresh tube. Make sure that all liquid is out.

Take filter and cut into 4 pieces and put into elution buffer with DNA and put the tube to water bath at 70-80oC over the course of 5 min.

The elute was transferred to a fresh tube (Elute 1) and filter was eluted again as above (Elute 2)

6. Precipitation

The elutes (1 and 2) were diluted with 200 μl H2O (each) before ethanol precipitation;

For precipitation: 2x6 μl glycogen (20 mg/ml) + 2x200 μl ammonium acetate (7.5 M) + 2x1 ml ethanol (100%) were incubated for 1 hour (or can leave overnight if no pellet is observed after centrifugation) at -80oC;

Centrifugation under 13,000 rpm at 4oC for 1 hour;

Carefully remove the supernatant without disturbing the pellet (S1 and S2 – keep supernatant just in case)

Washing the pellet with 75% ethanol solution (-20oC) (1000 μl) (add 1000 ul of ethanol and make sure the pellet came out; centrifuge for 5 min to let the pellet settle again; remove ethanol without disturbing the pellet leaving some liquid in the tube)

Repeat previous step one more time (2 washes of each pellet; WP1-1, WP1-2 for pellet from E1; WP2-1, WP2-2 for pellet from E2)

After the second wash carefully remove ethanol without disturbing the pellet with a pipette

Dry under the current of air in horizontal form for 2-3 minutes until it becomes transparent (maximum 5 minutes)

Resolve the pellet of Elute 1 in 50 μl pure water

After washing and drying the pellet from Elute 2, add resolved pellet from Elute 1 (50ul) into it and it is ready to do PCR.

7. PCR (dsDNA)

Mixture:

μl
DNA (random library), template	1
10*PCR buffer with MgCl2	5
dNTPs (2.5 mM)	4
DL-F (20 μM)	1
DL-R (20 μM)	1
Taq Polymerase (2.5 U)	0.5
dH2O	37.6
Total	50

PCR conditions:

95oC	5 min
95oC	30s
64oC	45s
72oC	45s
72oC	10min
4oC	Keep at

PCR product is about 80 bp

8. Check PCR product with 6% TBE gel

Line 1: 1 μl marker + 7 μl loading buffer

Line 2: 2 μl DNA library 6 1 μl loading buffer

Line 3: 3 μl product 1 + 5 μl loading buffer

Line 4: 3 μl product 2 + 5 μl loading buffer

μ 20x SYBR Green is added to each well

Gel running conditions: 200V

@@ Line 80: / Line 80: @@
 </ul>
-</body>
 <table id="toc" class="toc"><tr><td><div id="toctitle"><h2>Contents</h2></div>
 <ul>
@@ Line 87: / Line 87: @@
 </td></tr></table><script>if (window.showTocToggle) { var tocShowText = "show"; var tocHideText = "hide"; showTocToggle(); } </script>
 <h2> <span class="mw-headline" id="SELEX">SELEX procedure</span></h2>
-<ul><u><b>Materials:</b></u>
+<div><u><b>Materials:</b></u></div>
 <li>Binding buffer: 50mM Tris-HCl, pH 7.5, 25mM NaCL, 5 mM MgCl2, 10mM dithiothreitol (DTT);</li>
 <li>Wash buffer is same as binding buffer;</li>
 <li>Elution buffer: 0.4 M sodium acetate, 5mM EDTA, and 7M urea, pH 5.5;</li>
-<li>MF-Millipore membrane, mixed cellulose esters, hydrophilic, 0.45 μm, 13 mm. white, plain (HAWP filter, 0.45 μm, 13 mm diameter, Millipore) – 2 pcs;</li>
+<li>MF-Millipore membrane, mixed cellulose esters, hydrophilic, 0.45 μm, 13 mm. white, plain (HAWP filter, 0.45 μm,13 mm diameter, Millipore) – 2 pcs;</li>
 <li>Filter holder – 2 pcs</li>
-<li>ssDNAs library was dissolved in _______ μl sterile water, and concentration was 1 μg/ μl;</li>
+<li>ssDNAs library was dissolved in sterile water, and concentration was 1 μg/ μl;</li>
 <li>Target protein</li>
 <li>Petri dish – 1 pcs</li>
@@ Line 101: / Line 101: @@
 <li>Ethanol (100% and 75%)</li>
 <li>10*PCR buffer with MgCl2; dNTPs (2.5 mM); DL-F (20 μM); DL-R phophorylated (20 μM); Taq Polymerase; Lambda exonulcease</li>
-</ul>
-<ul><u>Equipments:</u>
+<div><u><b>Equipments:</b></u></div>
 <li>Thermocycler</li>
 <li>Rotator with variable speed (15 rpm) for 1 ml tubes</li>
 <li>Freezers (-20 and -80oC)</li>
 <li>Centrifuge – 13000rpm</li>
-</ul>
-<u>Buffers for SELEX</u>
+<u><b>Buffers for SELEX</b></u>
-     <ul>Binding buffer/Wash buffer:
+     <div><u>Binding buffer/Wash buffer:</u></div>
 <li>DTT- Dithiothreitol, Tris-HCl pH 7.5</li>
 <li>Tris-HCl (50mM) – 7.88g for 1L</li>
 <li>NaCl (25mM) – 1.461g for 1L</li>
 <li>MgCl (5mM) – 0.476g for 1L</li>
-<li>DTT (10mM) – 1.5425g for 1L</li></ul>
+<li>DTT (10mM) – 1.5425g for 1L</li>
-     <ul>Elution buffer:
+     <div><u>Elution buffer:</u></div>
 <li>Sodium acetate (0.4M) – 32.812g for 1L</li>
 <li>EDTA (5mM) – 1.86g for 1L</li>
 <li>Urea (7M) pH 5.5 – 420.42g for 1L</li>
-<li>Ammonium acetate (7.5M) – 578.7g for 1L/ 28.9g for 50ml</li></ul>
+<li>Ammonium acetate (7.5M) – 578.7g for 1L/ 28.9g for 50ml</li>
-<ol><b><u>Procedure</b></u>
+<b><u>Procedure</b></u>
-<li><ul><b>Pre-wetted</b></li>
+<div><b>1. Pre-wetted</b></div>
 <li>Soak membrane in sterile water for 1min and put membrane in the filter holder;</li>
 <li>To eliminate DNAs that bound to the membrane, DNAs (1 μg/μl) were passed three times prior to the selection cycle through pre-wetted nitrocellulose acetate membranes  in “Pop-top” filter holders;</li>
-</ul>
-<li><ul><b>DNA denature</b></li>
+<div><b>2. DNA denature</b></div>
 <li>For the first cycle of selection, 35.5 μl (1μg/ μl) DNAs were added in 114.5 μl binding buffer solution (volume 150 μl);</li>
 <li>And then were denatured at 95oC for 10 min;</li>
 <li>Then were allowed to cool in thermocycler to approximately 300C (about 1.5 hours);</li>
-</ul>
-<li><b>Binding</b></li>
+<div><b>3. Binding</b></div>
 Denatured DNAs (35.5 μl, 1μg/ μl DNAs + 114.5 μl binding buffer) + 50 μl target protein (87 μg/ml) were incubated at 15 rpm on a variable speed rotor for 1 hour and 45 min at room temperature.
-<li><ul><b>Washing</b></li>
+<div><b>4. Washing</b></div>
 <li>Soak membrane in wash buffer (the same as binding buffer) for appr. 1 min and then put the membrane in the filter holder;</li>
 <li>Aspirate the binding reaction mixture (DNAs+target) with a syringe with a needle</li>
 <li>Remove the needle and inject the binding reaction mixture into the filter holder with a membrane and then inject 1 ml wash buffer solution (the same as binding buffer) and let wash buffer pass through the membrane.</li>
-</ul>
-<li><ul><b>Elution</b></li>
+<div><b>5. Elution</b></div>
 <li>Heat 200ul elution buffer(0.4 M sodium acetate, 5 mM EDTA, 7M urea, pH 5.5) to 70-800C</li>
 <li>Inject 200 μl of elution buffer into the filter holder to elute DNA that was retained in the filter into a fresh tube. Make sure that all liquid is out.</li>
 <li>Take filter and cut into 4 pieces and put into elution buffer with DNA and put the tube to water bath at 70-80oC over the course of 5 min.</li>
 <li>The elute was transferred to a fresh tube (Elute 1) and filter was eluted again as above (Elute 2)</li>
-</ul>
-<li><ul><b>Precipitation</b></li>
+<div><b>6. Precipitation</b></div>
 <li>The elutes (1 and 2) were diluted with 200 μl H2O (each) before ethanol precipitation;</li>
 <li>For precipitation: 2x6 μl glycogen (20 mg/ml) + 2x200 μl ammonium acetate (7.5 M) + 2x1 ml ethanol (100%) were incubated for 1 hour (or can leave overnight if no pellet is observed after centrifugation) at -80oC;</li>
@@ Line 155: / Line 155: @@
 <li>Dry under the current of air in horizontal form for 2-3 minutes until it becomes transparent (maximum 5 minutes)</li>
 <li>Resolve the pellet of Elute 1 in 50 μl pure water</li>
-<li>After washing and drying the pellet from Elute 2, add resolved pellet from Elute 1 (50ul) into it and it is ready to do PCR.</li></ul>
+<li>After washing and drying the pellet from Elute 2, add resolved pellet from Elute 1 (50ul) into it and it is ready to do PCR.</li>
-<li><b>PCR (dsDNA)</b></li>
+<div><b>7. PCR (dsDNA)</b></div>
 <table border="1"> <b>Mixture:</b>
 	<tr><td>μl</td></tr>
@@ Line 182: / Line 182: @@
 <div>PCR product is about 80 bp</div>
-<li><b>Check PCR product with 6% TBE gel</b></li></ol>
+<div><b>8. Check PCR product with 6% TBE gel</b></div>
 <div>Line 1: 1 μl marker + 7 μl loading buffer</div>
 <div>Line 2: 2 μl DNA library 6 1 μl loading buffer</div>
@@ Line 191: / Line 191: @@
 <div>Gel running conditions: 200V</div></table>
+</body>
 </html>