Team:NU Kazakhstan/Protocols

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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

Cloning and transformation

Materials:

Resuspended DNA (Resuspend well in 10ul dH20, pipette up and down several times, let sit for a few minutes) Competent cells (50ul per transformation)

Ice (in ice bucket/container)

2ml tube (1 per a transformation')

42ºC water bath

SOC media (check for contamination!)

Petri dishes with LB agar and appropriate antibiotic (2 per transformation)

glass beads or spreader

37ºC incubator

10pg/ul RFP Control (pSB1A3 w/ BBa_J04450)

Procedure:

Start thawing the competent cells on ice.
Add 50 µL of thawed competent cells into pre-chilled 2ml tube, and another 50µL into a 2ml tube, labelled for your control.
Add 1 - 2 µL of the resuspended DNA to the 2ml tube. Pipet up and down a few times, gently. Make sure to keep the competent cells on ice.
Add 1 µL of the RFP Control to your control transformation.
Close tubes and incubate the cells on ice for 30 minutes.
Heat shock the cells by immersion in a pre-heated water bath at 42ºC for 60 seconds.
Incubate the cells on ice for 5 minutes.
Add 200 μl of SOC media (make sure that the broth does not contain antibiotics and is not contaminated) to each transformation
Incubate the cells at 37ºC for 2 hours while the tubes are rotating or shaking. Important: 2 hour recovery time helps in transformation efficiency, especially for plasmid backbones with antibiotic resistance other than ampicillin.
Label two petri dishes with LB agar and the appropriate antibiotic(s) with the part number, plasmid backbone, and antibiotic resistance. Plate 20 µl and 200 µl of the transformation onto the dishes, and spread. This helps ensure that you will be able to pick out a single colony.
For the control, label two petri dishes with LB agar (AMP). Plate 20 µl and 200 µl of the transformation onto the dishes, and spread.
Incubate the plates at 37ºC for 12-14 hours, making sure the agar side of the plate is up. If incubated for too long the antibiotics start to break down and un-transformed cells will begin to grow. This is especially true for ampicillin - because the resistance enzyme is excreted by the bacteria, and inactivates the antibiotic outside of the bacteria.
You can pick a single colony, make a glycerol stock, grow up a cell culture and miniprep.
Count the colonies on the 20 μl control plate and calculate your competent cell efficiency.

DOT ELISA for biotynilated CEA

Materials

Nitrocellulose membrane

Streptavidin-HRP

BCIP/NBT

Blocking buffer