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
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