1. How to determine the OD value of the primer?
Use a UV spectrophotometer to measure the absorbance of the solution at 260nm wavelength to quantify. Please pay attention to the use of UV spectrophotometer. The absorbance of the solution should be diluted to 0.2-0.8 during the measurement (absorbance is too high or too low will have a larger error). After the DNA dry powder is sufficiently shaken and dissolved with a certain volume of water, a part of the solution is diluted to 1 ml and the absorbance is measured in a 1 ml standard cuvette, which is the OD value of the measured volume, and then the OD value of the mother liquor can be calculated.
Example: You get a tube of dry powdered DNA, dissolve it in 1ml of water to make a mother liquor, take 50μL of the mother liquor to 1ml and measure the absorbance in a 1ml standard cuvette to 0.25, indicating that the 50μL contains 0.25OD of DNA, and That means that the original 1ml mother liquor contains 5OD DNA.
2. How to dissolve the primer?
Our synthesis report gives the amount of water added at a concentration of 100 μmol / L (ie 100 pmol / ul) per OD primer. You can add appropriate amount of nuclease-free double-distilled water (PH> 6.0) according to your experiment. ) Or TE buffer (PH 7.5-8.0), it is best to centrifuge at 3000-4000 rpm for 1 minute before opening the bottle to dissolve to prevent the primers from being lost when opening the cap.
3. How should synthetic primers be stored?
Undissolved primers are very stable and can be stored at -20 ° C for at least 1 year. Dissolved primers can be diluted to a storage solution of 100 μmoL / L in advance. Store several portions in a -20 ° C refrigerator for at least half a year ( Repeated freezing and thawing will reduce the service life). Before use, dilute the concentrated solution into a working solution (10 pmol / ml or 20 pmol / ml) and perform the experiment.
4. How to detect the purity of primers?
The convenient method in the laboratory is to use the PAGE method. Use a certain concentration of polyacrylamide gel with 7M urea for electrophoresis, <12 base primers use 20% gel, 12-60 base primers use 16% gel,> 60 bases Use 12% gel for primers, take about 0.2OD primer, dissolve with urea saturated solution or add dry urea powder to the primer solution until saturation, heat and denature before loading (95oC, 2mins). The purpose of adding urea is denaturation, and the second is to increase the specific gravity of the sample, which is easy to add. Carry out electrophoresis at 600V voltage. After a certain period of time (about 2-3 hours), strip the glue and use a fluorescent TLC plate to detect the band pattern under the UV lamp. There is no miscellaneous band under the main band, indicating that the purity is good (sometimes due to denaturation (Sufficiently, there may be a band above the main band, which is a primer secondary structure band).
5. Do the general synthetic primers have phosphate groups at the 5 and 3 ends?
No, both the 5 and 3 ends are -OH groups. If you need to add phosphoric acid group, please specify when ordering, in this case, you need to charge the phosphorylation fee.
6. What should I do if the synthesized primers have no purpose band during PCR reaction?
There are many reasons for the failure of the PCR reaction, which can be considered from the following aspects.
1) Are the primers and templates matched, and how much homology?
2) Whether the primer itself has a three-dimensional structure.
3) Can the reagents for PCR work properly?
4) Is the thermal cycler working properly?
5) Are the PCR reaction conditions appropriate?
If everything is normal and the problem cannot be solved, we can re-synthesize the primers for free.
7. After measuring the OD value of the primer, it is found that A260 / A280 <1.8, is the purity of the primer acceptable?
Since nucleic acids have strong absorption around 260nm, and proteins have strong absorption around 280nm, when extracting nucleic acids from the body, the ratio of A260 / A280 is often used to evaluate the purity of the nucleic acid (the ratio is between 1.8 and 2.0). This judgment is based on the sequence The result when the proportions of A, G, C, and T are almost the same. Synthetic DNA / RNA is different, and the sequence is very short (usually between 20 and 30 bases), where the proportions of various bases A, G, C, and T are very different. The molar extinction coefficient is different, so the primers composed of different bases have different A260 / A280 ratios. For example, when the content of C and T bases in the sequence is high, the ratio will be much lower than 1.8. Therefore, the ratio of A260 / A280 cannot be used to judge the purity of the primer.
8. How long can Shanghai Shenggong Company synthesize?
Due to the requirements of users and gene splicing, we have successfully synthesized many long fragments of about 100 bases in length. Because we can increase the amount of initial synthesis, increase the amount of reagents used for synthesis, and use PAGE for purification. If your experiment requires, we are willing to accept orders below 110 bases.
9. After cloning the PCR product and sequencing, it is found that the primer region does not match the synthetic sequence, what should I do?
We believe that most of these are errors introduced during PCR and cloning. In this case, please:
1) Can request us to re-synthesize primers for free.
2) Re-picking clones and sequencing, there is a possibility of finding the correct clone.
10. How to anneal two complementary single strands to form a double strand?
Dissolve the primers in annealing buffer (10 mM Tris, pH 7.5-8.0, 50 mM NaCl, 1 mM EDTA), mix the primers to be annealed equimolar, the total volume should not exceed 500 μl, heat to 95 ° C for 2 mins, and then slowly cool to room temperature (Less than 30 degrees). The annealed product can be placed at 4 degrees for use.
11. Using 3% Agarose gel electrophoresis to analyze the synthesized primers and found that there are many swimming bands, why?
Denaturing PAGE electrophoresis must be used for electrophoresis of primers. Because the primers are single-stranded DNA, it is easy to form a complex three-dimensional structure, so when Agarose electrophoresis, multiple bands are prone to appear, and it is impossible to quantify by Agarose electrophoresis.
12. Can the synthetic primers be quantified based on the brightness of the bands after EB staining after primer electrophoresis?
Can't. Because EB is colored by intercalating between the double helix of nucleic acid. The synthesized DNA molecule is single-stranded, and can only be stained by EB if it forms a partial hairpin loop structure or forms a partial double helix structure between the chains by folding back on itself. Due to the different sequences of different primers and the different ability to form a double helix, the staining ability is not the same, so the synthetic DNA cannot be quantified according to the brightness of the EB staining band.
13. How many PCR reactions can the primer of 2OD be used for?
Generally speaking, a primer with 2OD of about 20 bases can do at least 400 PCR reactions.
14. What impurities are contained in the crude product of DNA synthesis?
The crude product synthesized by the DNA synthesizer, in addition to the desired target DNA fragment, also contains the short target fragment generated during the synthesis reaction and the ammonium salt generated by the deprotection group. The primers provided by our company have all Short fragments are removed by purification and salts are removed by desalting.
15. Will the primer degrade when transported at room temperature?
It will not degrade. The dried primer can be stored stably for more than two weeks at room temperature. The general shipping time is usually 1-3 days, so the primers you receive will not degrade.
16. Why are the charges for long-chain primers higher than short-chain primers?
Generally, when synthesizing long-chain primers, the amount of reagents added is much more than that of short-chain primers, especially for primers larger than 90bp bases. Due to the increase in cost, the price is also higher.
Reasons for PCR DNA sequencing error 1. Taq enzyme cause
The company is now one of the world's largest synthetic DNA professional companies, synthesizing about 2100 primers for users around the world every day. Most of these large numbers of primers are used for PCR amplification. About five ten thousandths after DNA sequencing, errors were found in the primer part, and most of the errors were lost. Many users believe that this kind of error is caused by our company's synthesis error. In fact, this kind of error is caused by the inherent error probability of Taq enzyme and has nothing to do with synthesis. Please see the following diagram:
It can be clearly seen from the figure that the primer part is also amplified by Taq. Since the primer is also amplified, an error may occur. Will there be any mistakes in chemically synthesizing DNA? The answer is no. There are only two kinds of errors: one is that the base is replaced; the other is that the base is lost. The company's more than 80 DNA synthesizers are all products of ABI. ABI's DNA synthesizer is the most reliable in the world. I have not heard of ABI's DNA synthesizer when synthesizing a base (such as A), but it is wrong. If you add another base (G, C or T), the loss is even more unlikely. Because DNA synthesis is performed on a solid phase, the synthesis of each base includes steps such as deprotection (DMT), base addition, capping, and oxidation. If a certain base is not added to the extending Oligo due to various reasons (such as the solution in the bottle is gone, or the pipe is blocked), then the next reaction Capping will seal Oligo, so that the entire oligo synthesis is immediately stopped. It can be seen that the error in the primer part when sequencing the above PCR products is not a mistake in primer synthesis, but caused by the Taq enzyme.
2. Chemical reasons
During the synthesis process, if the DNA synthesis report provided by our company is correct, it indicates that the synthesis was successful. The possibility of base mutation caused by human factors can be completely ruled out. DNA synthesis experts Dr. Hecker and Dr. Rill made a comment on this (Error Analysis of Chemically Synthesized Polynucleotides Biotechniques, 1998. Feb. 24: 256-60), thinking that the chemically synthesized DNA fragments are more than natural DNA fragments Multiple base mutation probability. But its true chemical mechanism is not yet clear, but what is certain is that it is impossible to ensure that 100% of errors do not occur. Recently, Dr. Jacek Lubkowski published an article in Nucleic Acids Research (2002, Vol. 30, No. 10 e43) which also confirmed that chemically synthesized primers (non-human errors) will cause errors, and the longer the primer chain , The higher the probability of error. The original text is as follows: while this method is simple in principle, in practice numerous complications can lead to errors in the synthesis. To reduce the possibility of errors during oligonucleotide synthesis, the oligonucleotides should be rather short, yet they must still be long enough to provide stable priming overlaps.
Third, the solution:
1. It is recommended to use high-fidelity high-temperature polymerase, such as Super Pfu, Pfu, Taq plus, etc., the fidelity of which is about 10 times higher than Taq enzyme, which can effectively reduce errors.
2. Pick another clone for DNA sequencing. Generally speaking, it is less likely to make mistakes again.
3. Re-synthesize primers.
4. Send your clones with missing bases to our genetics department. We are responsible for point mutation of the missing bases to provide the correct sequence you need. However, because the customer ’s carrier is very complex and changeable, our commitment is to provide
Source of DNA damage
1.1 The bases fall off to form AP sites. Heat and acid can make purines and pyrimidines fall off from the ribose phosphate backbone of the DNA chain to form AP sites (Apurinic or Apyrimidinic site).
The glycosidic bond of alkylated guanine is unstable, and it is easy to fall off to form a baseless site on DNA.
1.2 Base changes â‘ Physical factors ionizing radiation can cause other substances to generate free radicals, which can cause base oxidation modification, peroxide formation, base ring destruction and shedding, etc.
Generally pyrimidine is more sensitive than purine.
â‘¡Chemical factors
a. Alkylating agent dimethyl sulfate, methyl methanesulfonate (MMS) and other alkylating agents can add alkyl to N or O of purine or pyrimidine to alkylate the base.
N7 of guanine and N3 of adenine are most vulnerable to attack, forming m7G and m3A alkylated purine bases, resulting in base mismatch during replication.
For example, guanine N7 will be paired with T after being alkylated, and as a result, GC will be converted to AT.
b. Base analogues
5-bromouracil (5-BU), 5-fluorouracil (5-FU), 2-aminoadenine (2-AP), etc. Their structure is similar to bases, and entering the cell can replace normal bases and participate in the DNA chain to interfere with DNA replication synthesis.
If the structure of 5-BU is similar to T, it is paired with A in the keto structure; it is more likely to be paired with the enol structure and G, which causes AT to be converted to GC during DNA replication.
c. Aflatoxin Aflatoxin B, 1,2-acetyl-aminofluorene, benzopyrene, acridine, etc. can be inserted into the base sequence, causing frame shift.
d. Nitrate and nitrite can make C deamination into U, and after replication can make GC on DNA into AT pairs.
â‘¢Spontaneous change and damage of bases
a. Isomerization of bases
The isomers of the four kinds of bases can be spontaneously interconverted (the interconversion between the enol form and the ketone form).
b. Deamination of bases The NH2 outside the base of the base may sometimes spontaneously fall off, causing C → U, A → hypoxanthine (I), G → xanthine (X), etc. During DNA replication, UA, IX, IC pairing leads to wrong DNA sequence of the progeny.
Demethylation of 5-methylcytosine produces T (causing a change in CG → TA), while C deamination produces U (it is usually removed or replaced by C).
â‘£ Oxygen free radicals damage cell metabolic byproducts O2-, H2O2, etc. will cause base damage, produce base modifications such as thymine glycol, hydroxymethyluracil, and cause base pairing errors.
1.3 Base insertion or deletion Acridine molecules are positively charged and flattened, which is easy to insert between the base planes of DNA, resulting in the deletion or insertion of a base during the replication or recombination process.
DNA polymerase slips during the replication process, especially the deletion or insertion of one or several bases in the segment of several consecutive same bases. Polymerase sliding on the template chain is easy to cause deletion, and sliding on the growth chain is easy to cause insertion.
Insertion or deletion will cause the reading frame to change.
1.4 Pyrimidine dimer
When DNA is irradiated with ultraviolet light, the adjacent pyrimidines on the DNA chain are covalently bonded into a dimer.
Two adjacent Ts; or two Cs; or both C and T can form cyclobutyl dimer; between two adjacent Ts is the easiest to form TT dimer.
1.5 DNA strand breaks Ionizing radiation can cause DNA strand breaks; both direct and indirect effects of radiation may cause deoxyribose damage or phosphodiester bond breaks and cause DNA strand breaks.
Alkylating agents can also break DNA strands; the oxygen on the phosphodiester bond of the DNA chain is easily alkylated, resulting in an unstable phosphotriester bond that hydrolyzes between sugar and phosphoric acid, breaking the DNA strand.
A double-strand break in a haploid cell is a lethal event.
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