At the beginning of the experiment, first explain some necessary steps of the experiment (note: the test methods used in almost every small experiment). Avoid too much confusion in the process of writing experimental reports! Moreover, these processes are not attended by every student, so they are written at the beginning of this experimental report.

Preparation of (1) 1% agarose gel

Weigh 1g agarose, put it in a conical flask, add 100mL 1×TAE buffer, and heat it in a microwave oven. When it is completely melted, take it out and shake it well.

(2) Preparation of rubber sheet

1. Seal the edges of both ends of the rubber sheet with adhesive plaster.

2. Put the rubber plate in a horizontal position and put away the sample comb.

3. Slowly pour the agarose gel cooled to about 60℃ into the rubber plate, taking care not to generate bubbles. The thickness of the glue is between 3 and 5 mm.

4. After the glue is solidified, take out the comb, remove the adhesive tape and put it in the electrophoresis tank (note: the direction of the comb is close to the negative electrode).

5. Add 1 × TAE buffer into the electrophoresis tank, and the buffer should be immersed in the gel.

(3) Add samples

1. Add an appropriate amount of 10 × loading buffer to the sample, so that the final concentration of the buffer is 1×.

2. Use a micropipette to add the sample added with buffer to the sample loading hole. Record the order and quantity of samples added. (Note: Do not let the sample overflow from the sample adding hole during the sample adding process.)

(4) electrophoresis

1. Connect the power supply of electrophoresis tank and electrophoresis instrument (note that DNA fragments move from negative electrode to positive electrode). The migration speed of DNA is proportional to the voltage. The maximum voltage shall not exceed 5V/cm.

2. When bromophenol blue dye moves to 2/3 of the gel, stop electrophoresis.

(5) dyeing

Immerse the gel after electrophoresis in ethidium bromide staining solution (glove operation).

(6) Observe the experimental results.

The stained gel was observed under ultraviolet lamp (360 nm or 254 nm), and orange fluorescence band appeared at DNA.

Experiment 1 Extraction of E.coli Genome

Experimental purpose

1. Learn and master the extraction method of bacterial genome.

Experimental principle

DNA is a kind of circular macromolecular DNA, and eukaryotic DNA exists in the nucleus in the form of chromosomes. Different species of organisms and different forms of cells (such as fungi, cultured cells, plant tissues and animal tissues) have different genome extraction methods, but the basic principle is similar, that is, DNA should be separated from protein, lipids and sugars, and DNA molecules should be kept intact. The general process of DNA extraction is to digest and decompose protein in a solution containing sodium dodecyl sulfate (SDS) and protease K, and then extract and separate protein with phenol and chloroform/isoamyl alcohol. The obtained DNA solution was precipitated with ethanol to separate DNA from the solution. The mechanism of SDS is that it can combine with protein to neutralize the electrical properties of protein, so that the non-valence bond of protein is destroyed and the secondary structure is lost, resulting in deformation and inactivation. The important feature of protease K is that it can still maintain high activity in the presence of SDS and EDTA. In the reaction system of extracting DNA after homogenate, SDS can destroy cell membrane and nuclear membrane by inactivating protein, so that tissue protein can be separated from DNA. Protease K can degrade protein into small peptides or amino acids, so that DNA molecules can be completely separated. CTAB (cetyltriethylammonium bromide) is a detergent, which can dissolve cell membrane and form a complex with nucleic acid. It is soluble in high salt solution (0.7 mol/L NaCl). When the salt concentration of the solution is reduced to a certain extent (0.3 mol/L NaCl), it will precipitate out of the solution, and CTAB- nucleic acid complex can be separated from protein and polysaccharide by centrifugation. Finally, DNA was precipitated by ethanol or isopropanol, and CTAB was dissolved by ethanol or isopropanol to remove it.

experimental materials

Escherichia coli DH5α bacterial solution

Experimental reagent

LB liquid medium, TE solution, 10%SDS, protease k, 5mol/L NaCl, CTAB/NaCl.

Solution, phenol/chloroform/isoamyl alcohol, isopropanol, 70% ethanol

Experimental instruments and equipment

Micro pipette, cryogenic centrifuge, water bath pot, eppendorf tube, constant temperature shaker.

experimental procedure

(1) Freeze and centrifuge 2mL of E.coli DH5α solution cultivated to logarithmic growth phase at 5000rpm 10min, and discard the supernatant;

(2) add 190μL TE suspension for precipitation, add 10%SDS, 1μL 20mg/mL protease K, mix well, and keep the temperature at 37℃1h; ;

(3) Add 30μL 5mol/L sodium chloride and mix well;

(4) Add 30μL CTAB/NaCl solution, mix well, and keep the temperature at 65℃ for 20min；;

(5) Add 300μL phenol/chloroform/isoamyl alcohol (25: 24: 1) for extraction, centrifuge at 5000rpm 10min, and transfer the supernatant to a clean centrifuge tube;

(6) add 300μL chloroform/isoamyl alcohol (24: 1) for extraction, and take the supernatant and move it to a clean test tube;

(7) add 300μL isopropyl alcohol, mix upside down, stand at room temperature for 65438±00min, and precipitate DNA；;

(8) centrifuge at 5000rpm for 65438+/-00 minutes, precipitate DNA, add 500μL70% ethanol, centrifuge at 5000 rpm for 65438+/-00 minutes, discard ethanol, and suck dry;

(9) Dissolve in 20μLTE, take 3μL for agarose gel electrophoresis verification, and keep the rest at -20℃.

Experimental results and analysis

109876m

As shown in the figure, No.8 is the experimental result of this group. Compared with maker, the first band is the normal E.coli genome, and two fuzzy bands can be seen in the middle, which may be broken genome fragments, and the brightest at the bottom is RNA. The fragmentation of the genome may be due to too intense operation. The electrophoresis bands in this group are still clear and tidy, and the irregular bands in other groups may be electrophoresis technical problems. The RNA content is high, so there is tailing phenomenon.

Experiment 2 preparation and verification of competent cells of Escherichia coli

Experimental purpose

1. Mastering ability