Determination of the amount of DNA fixed in ethanol

Summary

Determination of DNA content can be used to (1) perform gene fragment ligation experiments. (2) Perform plasmid concentration determination prior to transformation transfection.

Operation method

Determination of the amount of DNA fixed in ethanol

Principle

DNA and RNA have a very high absorption peak at 260nm, using this principle we measure their OD260 and extrapolate their concentration.

Materials and Instruments

Cell samples
PBS buffer 70% ethanol PI solution
Centrifuge Thermostat Sieve

Move

I. Determination of DNA content in cultured cells

Prepare single cell suspension in 200μ l of PBS buffer; add 2ml of pre-cooled 70% ethanol and store at 4°C. 1. Take 1-2×106 cells in PBS buffer (PH=7.2); take 1-2×106 cells in PBS buffer;

1. Take 1-2×106 cells from the single cell suspension in PBS (PH=7.2) buffer. 2;

2. centrifuge the cells at 300g for 5 min, discard the supernatant, and repeat twice. 3. resuspend the cells in 0.5 μmol/L;

3. Resuspend cells in 0.5 ml of PBS buffer. 4;

4. Place the cell suspension in 2--3 ml of cold 70% ethanol, mix well, and store at 4°C for at least 30 minutes. It can be stored at 4℃ for 2--3 weeks.

2. Determination of DNA content of fresh tissues

1. 200mg of wet weight tissue should be used to make single cell suspension by mechanical method. 2. 500g should be centrifuged for 5 min;

2. centrifuge at 500g for 5 minutes. 3;

3. Discard the supernatant and resuspend it in 10ml of staining and decontaminating agent. 4;

4. re-filter through a 200-mesh sieve or 70-80 μm sieve;

5. test on the machine.

Determination of DNA content in paraffin-embedded tissue sections.

1. Take 50 μm thick slices from paraffin-embedded sections, 2--3 slices, and make single-cell suspension. 2;

2. Wash with PBS buffer, centrifuge at 500g for 5 minutes and discard the supernatant. 3;

3. Add 1 ml of PI solution and protect from light for 30 minutes at room temperature. 4;

4. Adjust the cell concentration to 1×106/ml. 5;

5. Test on the machine.

Caveat

1. 1--3% paraformaldehyde can be used as fixative according to the requirements of the experiment.

2. ethanol should be pre-cooled to 0--4℃ when ethanol is used as fixative; 3. the fixative should be slowly dripped into the cell suspension so that the concentration of fixative increases slowly.

3. When cells are fixed, the fixative should be slowly dripped into the cell suspension to increase the concentration of the fixative and shaken continuously to avoid the cells from forming clumps (especially when fixed with ethanol).

Common Problems

If you want to test the purity of the sample, then you have to measure the OD value at 280nm again and calculate the ratio of the two, if the ratio is close to 2, it means that the sample is pure.

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