Protocols

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Sand Pack Test

Materials:

1. A type of surfactant
2. Petroleum or sunflower oil
3. Soil sample
4. Tap water
5. Plastic bottles
6. Frame

Investigating the optimal concentration:

1. Prepare plastic bottles and place them in the frame.
2. Weigh about 200g of soil sample, and fill them into the plastic bottle.
3. Measure 20ml of oil with a measuring cylinder, and add them to each soil sample.
4. Wait for 2hrs to allow the oil to combine with the soil.
5. Prepare surfactant solutions with different concentrations.
6. Add 20ml of surfactant solutions to the oil.
7. Place a cup under each bottle. Wait for 2hrs for the emulsion to occur.
8. Carefully cover the cups and freeze them in -20℃.
9. After the liquid freezes, separate the ice and soil residue from the remaining liquid by filtering. The volume of liquid should be equal to the volume of oil eluded from the soil. Record it in the table below.
10. Calculate the percentage elusion with (V/20ml)*100%.

THERE SHOULD BE A PICTURE OF THE TABLE FOR DATA

OEPCR(Overlap Extension PCR)

Materials:

1. Template for the left and right fragment
2. Primers A,B,C,D.
3. 2x Pfu mastermix
4. DNA purification kit

OEPCR Steps:

1. Amplify desired DNA fragments on left and right template with AB and CD primer pairs correspondingly. The annealing temperature is set to 55°C. When designing the primers, B is made to be complementary to C, with 30 bases overlapping with ends of the left and right fragments to be joined. (Make Tm be as close to 60°C as possible.)
2. Directly purify PCR product with a kit. Elute DNA with ddH2O.
3. Set a new PCR reaction with specific requirements: equimolar amount of left and right fragment PCR product from step 1, combined volume of the fragments should be 1/2 to 3/4 of the total volume, no new primers is added, annealing temperature is set to 60°C. Run PCR for 15 cycles.
4. Add primers AD to the previous PCR product and set annealing temperature to 72°C and run PCR for another 15-20 cycles.
5. Electrophorese, and cut gels of correct product length and recycle OEPCR product.

(If the amount of OEPCR product is not enough for later experiments, consider amplifying the product with AD primers, but be aware of nonspecific amplification results!)