Direct sequencing is a method to further study CpG island methylation based on MSP. Sulfite deamidates unmethylated cytosine in DNA and turns it into uracil, while methylated cytosine remains unchanged. After PCR amplification (CpG should be avoided as much as possible in primer design to avoid being affected by methylation factors), uracil was completely converted into thymine. Finally, the PCR products were sequenced. Compared with the untreated sequence, it can be judged whether CpG site is methylated or not. This method is highly reliable and accurate, and can determine the methylation status of each CpG site in the target fragment. It has unparalleled advantages in finding meaningful key CpG sites. Sequencing method takes a sequence without CpG point on both sides of CpG island as primer pairing region. Therefore, methylated and unmethylated target sequences can be amplified simultaneously. Its disadvantage is that it takes time and money. In order to obtain reliable data, at least 10 clones need to be sequenced. It needs a lot of cloning and plasmid extraction and sequencing, and the process is cumbersome and expensive.
The first part is the extraction of genomic DNA.
There is no suspense in this step. You can buy a set of DNA extraction tools for cells or tissues. If the laboratory conditions are ripe, you can extract it yourself. DNA is relatively stable. As long as violence is not used in the operation, the proposed genomic DNA should be complete.
This step focuses on the purity of DNA, that is, it is very important to reduce or avoid the pollution of RNA and protein, so protease K and RNase should be used to remove it in the extraction process.
Use the details of both:
1: protease k can be prepared into 20 mg/ml with sterilized double distilled water;
2.RNase must be prepared into RNase without DNase, that is, after RNase is purchased in the market, it is prepared into 10mg/ml. Otherwise, the possible consequence is that not only is there no 2:RNA, but DNA is also digested. Both were stored at -20℃.
There are two ways to verify the purity of the extracted DNA:
1: calculate OD ratio by ultraviolet spectrophotometer;
2: 1%- 1.5% agarose gel electrophoresis.
I prefer the second method, which can completely clarify the purity of the proposed genomic DNA and estimate its concentration according to the labeled sample size for the next revision.
The second part is the modification of genomic DNA by sodium bisulfite.
Unless otherwise specified, all DDW are sterilized by high pressure steam.
1: dilute about 2 micrograms of DNA to 50 microliters; Add DDW； into 1.5mlEP tube;
2: Add 5.5ul of newly prepared 3M NaOH；;
3: 42℃ water bath for 30 minutes; ;
Preparations during the water bath:
4: 10 mm hydroquinone, after water bath, add 30ul to the mixed solution (the solution turns pale yellow).
5: 3.6m sodium bisulfite (Sigma, S9000), preparation method: 1.88g sodium bisulfite diluted with DDW, titrated with 3M NaOH to PH 5.0, and constant volume to 5ml. Sodium bisulfite with such a large concentration is difficult to dissolve, but it will slowly dissolve after adding NaOH, which requires patience. The PH value must be exactly 5.0. After the water bath, 520 microliters were added to the above solution.
6: EP tube is wrapped in aluminum foil, protected from light, and the solution is gently inverted.
7: Add 200 ul paraffin oil to prevent water evaporation and limit oxidation.
8: 50℃ black water bath 16h.
Generally, this step starts at 4 pm, and it can be completed less than 5 pm if you are skilled. 16h water bath is just after 8 o'clock the next morning, which is very suitable.
Details of this step:
1: The amount of genomic DNA doesn't need to be very accurate, and it is better to have more than less, because it will be lost in the subsequent purification and recovery steps. This method can be modified to 4ug at most.
2. All reagents must be freshly prepared, so the technology of liquid preparation must pass, both fast and accurate.
3. The sodium bisulfite solution is strongly acidic, and the PH value must be adjusted to 5.0 with alkali, otherwise the improper PH value will affect the subsequent purification and absorption.
4. The best water bath is 16 hours. Although it can be as short as 8 hours, the later modification will be incomplete.
The third part is the purification and recovery of modified DNA
Unless otherwise specified, EP tubes are autoclaved.
1. Place the pipette head under the paraffin oil layer, first gently pressurize and discharge a small section of paraffin oil, and then suck the mixed liquid into a clean 1.5mlEP tube.
2. Next, use the Promega wizard to clean up the DNA purification and recovery system (Promega, A7280).
1)70℃ water bath preheating DDW；; ; Preparing 80% isopropanol;
2) Add Wizard DNA cleaning resin of 1ml Promega, gently invert and mix evenly, so that DNA and resin can be fully combined;
3) Because the kit is only equipped with a syringe and has no needle plug, it is very convenient to use if there is a vacuum negative pressure aspirator; If not, you need to bring your own 3ml-5ml syringe. After the syringe barrel is closely connected with the recovery column provided by the kit, the above mixture is transferred into the syringe with a pipette, and an EP tube of more than 2ml is placed under the column to receive the waste liquid. Add the needle plug, press gently and squeeze out the liquid. At this time, white resin deposits can be seen in the column.
4) After separating the syringe from the cartridge case, pull out the pin, then connect the syringe with the cartridge case, add 2ml of 80% isopropanol into the syringe, insert the pin, and gently press it to squeeze out the isopropanol. This is the washing step.
5) Separate the syringe from the cartridge, put the cartridge on a clean EP tube of 1.5ml, and centrifuge at 12000 rpm for 2 minutes to remove the residual isopropanol and dry the resin. At this time, the modified DNA is in a state of combining with resin.
6) Take out the chromatographic column and put it on another clean 1.5mlEP tube, add 50ml of preheated DDW into the pipette and leave it at room temperature for 5min.
7) Centrifuge 12000 for 20s, which is the elution step. At this time, the liquid in the EP tube is an eluted modified DNA solution with a final volume of 50ul.
3: Add 5.5 microliters of newly prepared 3M NaOH and leave it at room temperature for 65438 05 minutes.
4: Add 33ul 10M ammonium acetate to neutralize NaOH and make the PH value of the solution about 7.0.
5. Add 4U 10 mg/ml glycogen as a precipitation indicator, because it can produce precipitation after mixing with ethanol, which is convenient for identifying the position of the recovered material after centrifugation and preventing the recovered material from being sucked away when absorbing residual ethanol. In fact, it is hard to say how much these glycogen can play. However, there are domestic glycogen for sale, and the packaging is not big, which is very cheap. If you buy it, you should strictly follow the steps in the literature.
6: Add 270ul of ice anhydrous ethanol, put it at -20℃ and let it stand overnight. Some people think that the shortest settling time can be 2 hours, but I think it may take longer. When doing this step, it usually lasts until noon. If there are many samples, it may be better to leave them overnight, so the schedule can be relaxed and some other experiments can be done by the way. If you want to finish it on the same day, no problem, but I think it is best to settle it for a long time, at least 6 hours.
Centrifuge at 7: 4, 12000rpm for 30 minutes, pour out the supernatant and collect the precipitate. It's not necessary to absorb it completely.
8: Add 500ul 70% 70% ethanol, do not blow up the precipitate, just add ethanol. Gently tilt the EP tube, rotate it once, and centrifuge it again at 4 degrees 12000 rpm for 5 minutes. After centrifugation, pour off the supernatant, add the same amount of ethanol, and do it again. This is a washing step, twice.
9. Pour off the supernatant, centrifuge briefly at room temperature, separate the ethanol adhering to the wall to the bottom of the EP tube, carefully suck the residual liquid with a pipette, and dry for 5 minutes at room temperature, or add 20ul- 30ulDDW to dissolve the precipitate when it changes from opaque to translucent or transparent. So far, the purification and recovery of modified DNA have been completed, and the modified DNA solution can be used for further experiments.
10:-20℃ to store DNA solution.
Details of this step:
1: When using the syringe, the force must be even and light. If violence is used, the membrane in the column will be crushed and lose its function.
2. Ammonium acetate and glycogen do not need fresh preparation. Raw sugar should be stored at -20℃ after preparation, and ammonium acetate can be stored at room temperature because ammonium acetate with such concentration is very insoluble. Once placed at 4℃, a lot of solute will precipitate when taken out.
3. Isopropanol and 70% ethanol do not need fresh preparation, but if the dosage is large, it is convenient to prepare on site.
The key of this step is the combination of resin and DNA, and the importance of adjusting the PH value of sodium sulfite in the second part is emphasized again. Because the combination of resin and DNA needs a suitable PH value, if the previous step is not done well, the resin can not be well combined with DNA in this step, which will bring disastrous consequences, that is, DNA and liquid are squeezed out together, and there is actually no DNA when eluting.
The fourth part of the modified DNA was used for PCR.
There is no suspense in this step. I will mainly talk about a few thorny issues here:
1: Primer problem: I feel it is very difficult to design my own primers. I have designed several pairs of primers and tried them, but they all ended in failure. If there is enough time and there are not many relatively new genetic documents, it is no problem for me to design primers myself. If not, it is better to refer to the literature. Consult the literature with high SCI scores first, and then consult the literature of famous laboratories. It would be better to have it in China. You can contact the consultation directly. After the sequence is found, it must be compared with the sequence in Genbank to prevent individual base differences caused by typographical errors. Then you can search on Google to see if there are many users and the system conditions are the same. There are many users and the system conditions are the same, which shows that the repeatability is strong. I acted accordingly, too, and it went smoothly.
2.Taq enzyme problem: Some literatures use high-fidelity gold medal Taq (platinum), but I feel that as long as the system is correct and the conditions such as denaturation annealing are suitable, the general hot-start enzyme is still ok. I started using LA Taq from Takara, which is very easy to use. It is equipped with 10x LA buffer containing mg++. Sometimes you run out, you can temporarily use Takara's ordinary Taq enzyme. How to choose?
3.PCR conditions: denaturation is generally 95 degrees, 3m3min. I feel that the rest is based on the literature, and annealing can be tried in a small range according to your primer annealing temperature. There is little difference between general and literature reports. It's just a matter of the specificity of the amplified fragment. It is recommended to follow the literature.
4. It is best to choose imported EP tube for 4.PCR, with thin wall and uniform thickness, which can ensure that the rapid change of temperature can be transmitted to the reaction liquid in the tube in time, so that the system can really run at the set temperature.
5.PCR instrument: If it is done on a certain instrument, it is best to use this instrument all the time. Different instruments have different temperatures, but the EP tube must be closely combined with the jack in the instrument, leaving a gap, which I think will affect the temperature transfer.
This part is a bit verbose, just some immature personal experience. If you have any questions, please point them out and communicate. I came first today, and now I'm writing something, which is laborious and can't be done overnight. Please forgive me. I took a break and prepared to write the last part of the blue and white dot screening clone.
The fifth part is the gel recovery of PCR products.
This step is relatively simple. You can buy a gel PCR product recovery kit, which is made in China and has a reasonable price, such as Tiangen's products (second-hand). Please follow the instructions.
Some details:
1: The newly prepared electrophoresis solution was applied to agarose gel electrophoresis of PCR products. The gel concentration can be 1%-2%.
2. When recovering gel DNA, observe the band position under 300nm ultraviolet lamp, and cut the gel where the target fragment is located, as small as possible to ensure specificity.
3. The ultraviolet irradiation time should not be too long, otherwise it will damage DNA.
4. If the recovered DNA is not used immediately, it will be stored at -20℃ and will be very stable for several months.
The sixth part is the connection transformation of PCR products with T vector and the screening of blue and white spots.
1: Connecting T-vector (the kit of Promega is used in this experiment)
15ul system
T-easy 1ul
Ligase 1ul
2x buffer 7.5ul
DNA 5.5ul microliter
4 degrees, overnight.
2. Transformation of ligation products
1)-70 degrees Take out the competent bacteria from the refrigerator, melt them and put them on ice;
2) Add all the connecting products 15ul and ice for 30 minutes;
3)42 degrees 90 seconds;
4) 2 minutes on ice;
5)800ul LB medium;
6)280rpm, 37 degrees, shaking table for 45 minutes (drain the test tube and shake it evenly to ensure that the bacterial liquid is shaken evenly);
7)8000 rpm, 1 min; Remove the supernatant in the ultra-clean table, leaving100-150 ul;
8) coating: incubating at 37 degrees overnight; (Plate is solid LB medium containing ampicillin)
The first floor: X-gar 35ul
IPTG 25ul
Post-coating: suspension
After staying overnight, you can see many blue or white spots on the board. Taking white dots as an example, especially those around blue dots, the self-linking rate is low.
3. Take some white dots and plan them on the new board.
New circuit board: first painting: X-gar 35ul
IPTG 25ul
Then draw a partition on the bottom of the board and mark it. According to the need, it is generally no problem to make 50 clones on one board.
The needle selects a white spot and marks 2 lanes in the corresponding area on the board.
37 degrees, overnight in the incubator.
4. Contact the sequencing company to send sequencing. Generally, a clone is located in 35-45 yuan.
Details of this section:
1: The coating should be uniform, and Xgar and IPTG should be evenly distributed on the board;
2. Don't let the blue and white spots grow too full, otherwise it is easy to choose two clones at once.