Part 1 Extraction of Genomic DNA

This step can be achieved by purchasing DNA extraction kits for use by cells or tissues. If the laboratory conditions are mature, it is entirely possible to prepare your own reagents for extraction. DNA is relatively stable, as long as violence is not used during operation, the proposed genomic DNA should be complete. The focus of this step is on the purity of DNA, which is important to reduce or avoid contamination of RNA and proteins. Therefore, protease K and RNA enzymes need to be used in the extraction process to remove both.

Details of using both:

(1) Protease K can be prepared into 20mg/ml using sterilized double distilled water;

(2) RNA enzymes must be formulated as RNA enzymes without DNA enzymes, that is, after purchasing commercially available RNA enzymes, they must be reprocessed and formulated into 10mg/ml. Otherwise, the possible consequence is not only the absence of RNA, but also the digestion of DNA. Both are stored at -20 degrees Celsius.

There are two methods to verify the purity of extracted DNA:

(1) Calculate the OD ratio using a UV spectrophotometer;

(2) 1% -1.5% agarose gel electrophoresis.

It is suggested that agarose gel electrophoresis be used for detection: agarose gel electrophoresis can completely determine the purity of the genomic DNA and estimate its concentration according to Marker's loading amount for further modification.

Part 2 Sodium Bisulfite Modified Genomic DNA

Double distilled water (DDW) is sterilized by high-pressure steam.

(1) Dilute approximately 2ug of DNA in a 1.5ml EP tube with DDW to 50ul;

(2) Add 5.5ul of freshly prepared 3M NaOH;

(3) 42 ℃ water bath for 30 minutes;

Preparation during water bath:

(4) 10mM hydroquinone (hydroquinone), add 30ul to the mixture after the above water bath; (The solution turns pale yellow)

(5) 3.6M sodium bisulfite (Sigma, S9000), preparation method: Dilute 1.88g of sodium bisulfite with DDW and titrate the solution with 3M NaOH to pH 5.0, with a final volume of 5ml. Such a high concentration of sodium bisulfite is difficult to dissolve, but it will slowly dissolve after adding NaOH. Patience is needed. The pH must be accurate to 5.0. Add 520ul to the solution after the above water bath.

(6) Wrap the EP tube with aluminum foil, avoid light, gently invert and mix the solution.

(7) Add 200 ul of paraffin oil to prevent water evaporation and limit oxidation.

(8) 50 ℃ dark water bath for 16 hours.

Attention:

(1) The amount of genomic DNA does not need to be very precise, it is better to have more than less, as there may be losses in the purification and recovery steps in the future, and this method can modify up to 4ug.

(2) All reagents must be freshly prepared, so the preparation technique must be up to standard, both fast and accurate.

(3) Sodium bisulfite solution is strongly acidic, so it is necessary to adjust the pH to 5.0 with a base, otherwise an inappropriate pH will affect subsequent purification and absorption.

(4) It is best to take a water bath for 16 hours, although it can be as short as 8 hours, the latter modification may be incomplete.

Part 3: Purification and Recovery of Modified DNA

The EP tube is sterilized by high-pressure steam.

(1) Insert the pipette tip under the paraffin oil layer, gently pressurize to discharge a small section of paraffin oil, and then suck the mixed liquid into a clean 1.5ml EP tube.

(2) Purify and recover DNA after modification according to the operation requirements of the reagent kit

Part 4 Modified DNA for PCR

Follow the normal PCR operation, but pay attention to the following points:

(1) Primer issue: If there is enough time and the gene being developed is relatively new, with limited literature, design primers yourself. If time is tight, you can choose references. Firstly, consult literature with high SCI scores, followed by literature from well-known laboratories. If there is research done in China, you can directly contact for consultation. After finding the sequence, it is necessary to compare it with the sequence in Genbank to prevent individual base differences caused by printing errors. Then search again to see if there are many people using it and if the system conditions are the same. The use of multiple people and the same system conditions indicate strong repeatability.

(2) Taq enzyme problem: If the system is correct and the conditions for denaturation annealing are appropriate, a typical hot start enzyme can be used.

(3) PCR conditions: Denaturation is generally selected at 95 degrees Celsius for 3 minutes. According to the literature, annealing can be attempted within a small range based on the annealing temperature of your primer. Generally, there is not much difference from literature reports. It's just a matter of amplification fragment specificity. Suggest based on literature.

(4) It is best to choose imported EP tubes for PCR, with thin walls and uniform thickness, to ensure that rapid temperature changes can be transmitted to the reaction solution inside the tube in a timely manner, allowing the system to truly operate at the set temperature.

(5) PCR instrument: If it is produced on a certain instrument, it is best to continue using this instrument. Different instruments have different temperaments, but the EP tube must be tightly integrated with the socket inside the instrument, leaving gaps that can affect temperature transfer.

Part V gel recovery of PCR products

You can use the gel PCR product recovery kit to operate the cut glue according to the instructions.

Several details:

(1) For agarose gel electrophoresis of PCR products, the newly prepared electrophoresis solution should be used. The concentration of gel is 1% -2%.

(2) When recovering gel DNA, observe the band position under the 300nm ultraviolet lamp, cut the gel where the target segment is located, as small as possible, to ensure specificity.

(3) The UV irradiation time should not be too long, otherwise it may cause damage to DNA.

(4) If the recycled DNA is not immediately used and stored at -20 degrees Celsius, it is very stable for several months.

Part 6: Connection and Transformation of PCR Products with T Vector, and Screening of Blue and White Spot

(1) Connect T carrier (Promega's reagent kit)

15ul system: T-easy 1ul; Ligase 1ul; 2xbuffer 7.5ul; DNA 5.5ul, 4 degrees, overnight.

(2) Conversion of connecting products

-Remove receptive bacteria from a 70 degree refrigerator, melt them and place them on ice;

Add 15ul of the connecting product and let it sit on ice for 30 minutes;

42 degrees, 90 seconds;

On ice for 2 minutes;

800ul LB culture medium;

280rpm, 37 degrees, shaker for 45 minutes;

8000rpm, 1 minute; Remove the supernatant from the ultra clean table and leave 100-150ul;

Coating plate: Incubate overnight at 37 degrees Celsius; (The plate is a solid LB culture medium containing ampicillin)

First applied: X-gar 35ul, IPTG 25ul

Post coating: suspension

After staying overnight, many blue or white spots grow on the visible board. Take the white spots, especially the white spots around the blue spots, where the self connection rate is relatively low.

(3) Take the white spots and sow them on a new board

New board: First applied: X-gar 35ul, IPTG 25ul

Then draw zones at the bottom of the board and mark them. As needed, it is generally not a problem to make 50 clones on a single board.

Pick two white spots with a needle and mark them in the corresponding area on the board.

37 degrees, incubate overnight.

(4) Contact the sequencing company to send the sequencing.

The details of this section:

The coating should be uniform, ensuring that Xgar and IPTG are evenly distributed on the surface of the board;

Don't let the blue and white spots grow too full, otherwise when selecting clones, it's easy to pick two at once.