The best insertion fragment: vector ratio needs to be determined by experiments. 1: 1 (insert: vector) is often the best ratio, and the molar ratio of 1: 8 or 8: 1 is also acceptable. The ratio range should be determined. For ligation, 5ul 2X ligase, 50ng plasmid DNA, 1Weiss unit T4 ligase, and insert *** 10ul. Keep the temperature at room temperature 65438 0 hours, or stay overnight at 4℃. At these two temperatures, the carrier lacking T- projection will connect itself and produce blue dots. Keeping the temperature 1 hour at room temperature can meet most cloning requirements. In order to improve the connection efficiency, it is necessary to stay overnight at 4℃.
2) 2) Do PCR products need gel purification?
For example, gel analysis has only one band, and gel purification is not needed. If other bands can be seen, it may be that the dimer volume is tired of a large number of primers. A small amount of primer dimer also has a high molar number, which will produce a high proportion of clones with primer dimer, rather than the target insert. So gel purification is needed before cloning.
3) If the target fragment is not recovered, what control experiment is needed?
A) coating untransformed competent cells.
If there is a colony, it means that ampicillin is ineffective, or the plasmid with ampicillin resistance is contaminated, or the colony with ampicillin resistance is produced.
B) Transform the complete plasmid, calculate the colony growth number, and determine the transformation efficiency.
For example, dilute 1ug/ul plasmid 1: 100, and transform 100ul competent cells with 1ul. After being diluted to 1000ul with SOC, spread 100ul on the board. After overnight culture, 1000 colonies were produced. The conversion rate is: the total number of colonies produced and the total number of DNA diffusion.
The total amount of planking DNA is the amount used in the transformation reaction divided by the dilution multiple. Specifically, 10ng DNA was transformed, diluted to 1000u with SOC, containing 10 ng DNA, spread with110, and * * used 1 ng DNA. The conversion rate is:
1000 clone x103 ng/plank1ng DNA ug =106 cfu/ug.
PGEM-T was transformed with 108cfu/ ug competent cells.
If there are no colonies or few colonies, the transformation rate of competent cells is too low.
C) Using pGEM-T positive control or PCR products to produce >: 20-40 blue spots (using designated step 108cfu/ug competent cells), indicating that the vector lost T, which may be that ligase contaminated nuclease. T4DNA ligase (M 180 1, M 1804, M 1794) has good quality standard and no nuclease pollution, so T4 DNA ligase from other sources should not be replaced.
D) transforming high-frequency competent cells (108cfu/ug) with pGEM-T or pGEM-T Easy vector, and obtaining 100 colonies according to the specified experimental steps, of which 60% should be white spots, such as >: 20-40 blue spots, with no colonies or few colonies and connection problems.
4) The result of the control experiment is good, but the target fragment is not recovered. What's wrong with the experiment
A) The ligation can be maintained at room temperature for 65438 0 hours, which can satisfy most clones. In order to improve efficiency, it is necessary to stay overnight at 4℃.
B) The inserted fragment is contaminated, resulting in deletion of 3'-T or inhibition of ligation and transformation. Therefore, the inserted fragment was mixed with pGEM-T positive control and then ligated. If the colony number of the control is reduced, the inserted fragment needs to be purified or re-prepared. If a large number of blue dots are produced, it means that the inserted fragment is contaminated by nuclease, which makes pGEM-T or pGEM-T easy to delete the vector 3'-T.
C) The inserted fragment is not suitable for connection. Due to excessive ultraviolet irradiation, gel-purified inserts sometimes appear. Excessive ultraviolet radiation will produce pyrimidine dimer, which is not conducive to connection, and DNA must be purified again.
D) There is no A at the end of the amplification product of heat-resistant DNA polymerase with repair function, which is needed for cloning pGEM-T or pGEM-T Easy vector. Adding Taq DNA polymerase and nucleotide can add one at the end. Please refer to the technical data of pGEM-T pGEM-T Easy carrier (TM042) for details.
E) Highly repetitive sequences may be unstable, leading to deletion and rearrangement during amplification. If it is found that inserts are often deleted and rearranged, recombinant defective Escherichia coli strains, such as SURE cells, should be used.
PCR reaction system and reaction conditions
Standard PCR reaction system:
10× amplification buffer 10ul
Each of the four dNTP mixtures was 200 μ mol/L.
Each primer is 10 ~ 100 pmol.
Template DNA 0. 1 ~ 2ug
Taq DNA polymerase 2.5u
Mg2+ 1.5 mmol/L.
Add double or triple distilled water to 100 microliter.
Five elements of PCR reaction: There are five main substances involved in PCR reaction, namely primer, enzyme, dNTP, template and Mg2+.
Primers: Primers are the key to the specific reaction of PCR, and the specificity of PCR products depends on the complementary degree of primers and template DNA. Theoretically, as long as the sequence of any template DNA is known, complementary oligonucleotide chains can be designed as primers to amplify the template DNA in vitro by PCR. ① Primer length: 15-30bp, generally around 20bp.
② The primer amplification span: 200-500bp is appropriate, and the fragment of 10kb can be amplified under certain conditions.
③ Primer library: refer to the basic principles of primer design.
(4) Avoid the secondary structure in the primers, and avoid the complementarity between the two primers, especially the complementarity at the 3' end, otherwise primer dimer will be formed, resulting in non-specific amplification bands.
⑤ The bases at the 3' end of the primer, especially the last base and the penultimate base, should be strictly matched to avoid the failure of PCR caused by the mismatch of the terminal bases.
⑥ There is or can be a suitable restriction site in the primer, and it is better to have a suitable restriction site in the amplified target sequence, which is very beneficial to restriction analysis or molecular cloning.
⑦ Primer specificity: Primers should have no obvious homology with other sequences in the nucleic acid sequence database. Primer quantity: the concentration of each primer is 0. 1 ~ 1 umol or 10 ~ 100 pmol, and the lowest primer quantity is better to produce the required results. Higher primer concentration will lead to mismatch and nonspecific amplification, and increase the chance of dimer formation between primers. At present, there are two kinds of Taq DNA polymerase available, one is a natural enzyme purified from hydrothermal Bacillus, and the other is a genetic engineering enzyme synthesized by Escherichia coli. It takes about 2.5U of enzyme to catalyze a typical PCR reaction (when the total reaction volume is 100ul). Too high concentration will cause nonspecific amplification, and too low concentration will reduce the amount of synthetic products.
Quality and concentration of dNTP
The quality and concentration of dNTP are closely related to the efficiency of PCR amplification. DNTP powder is granular, and it will lose its biological activity if it is not stored properly. DNTP solution is acidic and should be prepared with 1M NaOH or 1M Tris after high concentration. Adjust the PH value of HCL buffer to 7.0 ~ 7.5, subpackage a small amount and freeze at -20℃. Repeated freezing and thawing will degrade dNTP. In the PCR reaction, dNTP should be 50 ~ 200 μm ol/L, especially the concentration of four dNTP should be equal (equimolar preparation). If any one of them is different from the others (higher or lower), it will cause mismatch. Too low concentration will reduce the yield of PCR products. DNTP can combine with Mg2+ and reduce the concentration of free Mg2+.
Template (target gene) nucleic acid The quantity and purification degree of template nucleic acid is one of the key links in the success or failure of PCR. Traditional DNA purification methods usually use SDS and protease K to digest and treat samples. The main function of SDS is to dissolve the lipid and protein on the cell membrane, so as to dissolve the membrane protein, destroy the cell membrane and free the nuclear protein in the cell. SDS can also combine with protein for precipitation. Protease K can hydrolyze and digest protein, especially histones bound to DNA, then extract protein and other cell components with organic solvents phenol and chloroform, and precipitate nucleic acids with ethanol or isopropanol. The extracted nucleic acid can be used as a template for PCR reaction. In general clinical samples, a quick and simple method can be used to dissolve cells, crack pathogens and digest protein of chromosomes, so that the target gene can be freed and directly used for PCR amplification. The extraction of RNA template usually adopts guanidine isothiocyanate or protease K method to prevent RNase from degrading RNA. Mg2+ concentration has a significant effect on the specificity and yield of PCR amplification. In general PCR reaction, when the concentration of various dNTP is 200 μm ol/L, the appropriate concentration of Mg2+ is1.5 ~ 2.0 mmol/L. If the concentration of Mg2+is too high, the specificity of the reaction will be reduced and nonspecific amplification will occur. If the concentration is too low, the activity of Taq DNA polymerase will decrease, and the reaction products will also decrease. The reaction conditions of PCR are temperature, time and cycle times.
Setting of temperature and time: based on the principle of PCR, set three temperature points: denaturation-annealing-extension. In the standard reaction, the three-temperature point method is used. Double-stranded DNA denatures at 90 ~ 95℃, and then rapidly cools to 40 ~ 60℃. After annealing, the primer binds to the target sequence, and then it is quickly heated to 70 ~ 75℃. Under the action of Taq DNA polymerase, the primer chain extends along the template. For short target genes (when the length is 100 ~ 300 BP), the double temperature point method can be used, and annealing and extension temperature can be combined in addition to denaturation temperature. Generally, it is denatured at 94℃ and annealed at about 65℃ (at this temperature, Taq DNA enzyme still has high catalytic activity).
① denaturation temperature and time: low denaturation temperature and incomplete melting are the main reasons for PCR failure. Generally speaking, the time from 93℃ to 94℃ is enough to denature the template DNA. If it is lower than 93℃, the time needs to be extended, but the temperature should not be too high, because the high temperature environment has an impact on the activity of the enzyme. If the target gene template or PCR product cannot be completely denatured at this step, PCR will fail.
② Annealing (renaturation) temperature and time: Annealing temperature is an important factor affecting the specificity of PCR. After denaturation, the temperature is quickly reduced to 40℃ ~ 60℃, so that the primer can be combined with the template. Because template DNA is much more complicated than primers, the probability of collision and binding between primers and templates is much higher than that between complementary chains of templates. The annealing temperature and time depend on the length of primer, base composition and concentration, and the length of target sequence. For primers with 20 nucleotides and 50% G+C content, 55℃ is the ideal starting point for selecting the best annealing temperature. The renaturation temperature of primers can help to select the appropriate temperature by the following formula:
Tm value (melting temperature) =4(G+C)+2(A+T)
Renaturation temperature =Tm value -(5 ~ 10℃)
Within the allowable range of Tm value, choosing a higher renaturation temperature can greatly reduce the nonspecific binding between primers and templates and improve the specificity of PCR reaction. The renaturation time is generally 30 ~ 60 seconds, which is enough to completely combine the primer and the template.
③ extension temperature and time: biological activity of Taq DNA polymerase;
70 ~ 80℃ 150 nucleotides/second/enzyme molecule
60 nucleotides/sec/enzyme molecule at 70℃
24 nucleotides/sec/enzyme molecule at 55℃
When the temperature is higher than 90℃, DNA synthesis is almost impossible.
The extension temperature of PCR reaction is generally 70 ~ 75℃, and the common temperature is 72℃. Too high extension temperature is not conducive to the combination of primers and templates. The time of PCR extension reaction depends on the length of the fragment to be amplified. Generally, for DNA fragments within 1Kb, the extension time of 1min is enough. The target sequence of 3 ~ 4 KB takes 3 ~ 4min；; The amplification of 10Kb needs to be extended to 15min. Excessive extension will lead to the appearance of non-specific amplification bands. For the amplification of low concentration template, the extension time is slightly longer. There are four PCR laboratories as required, namely 1 reagent storage and preparation area, 2 sample preparation area, 3 amplification reaction mixture preparation and amplification area, and 4 amplification product analysis area. See figure 1 for the layout. Figure 1 Regional Settings of Four PCR Laboratories
If a fully automatic analyzer is used, regions can be merged appropriately. We suggest that the amplification area and product analysis area be merged into three amplification detection areas, namely * * *. See Figure 2 for the layout.
Fig. 2 regional settings of three PCR laboratories
According to the requirements of the Ministry of Health, access to each work area must strictly follow a single direction, that is, only from the reagent storage and preparation area? Specimen preparation area amplification reaction mixture preparation and amplification area? Expand product analysis area to avoid cross-contamination. Appropriate ventilation equipment must be installed in each work area to ensure that the air flow direction is single. The walls, floors and office supplies in the work area must be made of materials resistant to disinfectants. The work area must be clearly marked to avoid the confusion of equipment and articles in different work areas. Each work area must be equipped with exhaust fans, air conditioners, corrosion-resistant floors and countertops, lockers, shoe cabinets, special office supplies, special work clothes, work shoes, mobile ultraviolet lamps, etc. To ensure safety and health needs.
According to the basic setting standards of clinical gene amplification laboratory of the Ministry of Health, the required instruments and equipment in each work area are as follows:
Reagent storage and preparation area
Refrigerators at 1.2-8℃ and-15℃
2. Agitator
3. Micro sampler (covering1-1000μ l)
4. Move the UV lamp (near the workbench)
5. Consumables: disposable gloves, disposable absorbent paper, high-pressure resistant centrifugal tube, sampler suction head (with filter element).
6. Special work clothes and work shoes
7. Special office supplies
Two specimen preparation areas
1.2-8℃ refrigerator, -20℃ or -80℃ refrigerator
2. High-speed tabletop freezing centrifuge
3. Agitator
4. Water bath box or heating module
5. Micro-sampler (covering 1- 1000 μ l)
6. Movable ultraviolet lamp (near the workbench)
7. Purification table
8. Consumables: the same as those in the "reagent storage and preparation area"
9. Special work clothes and work shoes
10. Special office supplies
If you need to process macromolecular DNA, you should have an ultrasonic water bath instrument.
Three gene amplification regions and product analysis regions.
1. Fully automatic quantitative nucleic acid amplification instrument (including computer and printer)
2. Micro sampler (covering1-1000μ l)
3. Movable ultraviolet lamp (near the workbench)
4. Consumables: the same as those in the "reagent storage and preparation area"
5. Special work clothes and work shoes
6. Special office supplies
Each work area must be clearly marked to avoid confusion of equipment and articles in different work areas. Work clothes with different colors or obvious distinguishing marks should be used in different work areas for identification. When workers leave the work area, they are not allowed to take out specific work clothes for each area.
3. Laboratory quality management
The staff of PCR laboratory must attend the clinical gene amplification training course organized by the Ministry of Health or the provincial clinical laboratory center, and hold relevant certificates. PCR laboratories have passed the acceptance test, and at least two laboratories must hold "Clinical Gene Testing Certificate". PCR laboratory must establish a series of quality management documents such as strict laboratory management system and standard operating procedures (SOP) to ensure that the daily operation of the laboratory meets the requirements of the Ministry of Health, the accuracy of test results, the hygiene and safety of the laboratory and the long-term stable operation of the laboratory. 1. touchdown PCR: the temperature gradually decreased in the first few cycles.
2. Reverse Transcription PCR(RT-PCR): The DNA obtained by using mRNA reverse transcription as a template does not contain introns (meaningless paragraphs in genes), which is a common method in molecular cloning technology.
3. Hot-start PCR: react with nucleic acid polymerase activated by high heat to reduce non-specific products.
4. Real-time PCR: quantitative detection with fluorescent probes or dyes during PCR, also known as quantitative PCR.
5. Nested PCR: First, amplify several cycles with low specificity primers to increase the number of templates, and then amplify with high specificity primers.
6. Multiplex PCR: Use multiple sets of primers in the same test tube.
7. Recombination PCR: The PCR product was diluted 10 times, and then put into the original concentration of primers and dNTP for three times to eliminate the heterodimers in the product.
8.dsRNA replicon: High fidelity DNA polymerase, T7RNA polymerase and PHi6RNA replicon are used together; Transcribed from double-stranded DNA into corresponding double-stranded RNA(dsRNA). It can be applied to the experimental operation of RNAi.
9. Cold PCR (Co-amplification atlas transformation temperature-PCR): a PCR application technology used to detect mutations or special alleles.