Frequently asked questions about primer synthesis

Q1. How are primers synthesized?

At present, the primer synthesis basically adopts the solid-phase phosphoramidite triester method. There are many kinds of DNA synthesizers. No matter what kind of machine is used for synthesis, the principle of synthesis is the same. The main differences are the level of synthesis yield, the amount of reagent consumption and the time spent in a single cycle.

(1) Deprotection: add Deblocking to remove the 5-OH protecting group DMT on the base to obtain free 5-OH;

(2) Coupling: adding activator and new base at the same time, the new base 5-OH is still protected by DMT, and the 3-terminal is activated to couple with the free 5-OH in solution;

(3) Blocking: A very small number of 5-OH in the coupling reaction did not participate in the reaction, and the reaction continued after terminating with blocking reagent;

(4) Oxidation: Add an oxidizing agent to form a more stable nucleoside phosphate from nucleoside phosphite.

Q2. How to deal with primer synthesis?

Cleavage and deprotection: chemically cleave the synthesized oligonucleotide chain from the support. Fresh concentrated ammonia is often used to cleave the ester bond between CPG and the initial nucleoside. The cleaved oligonucleotide has a free 3 'hydroxyl group.

Purification: The method of purification is selected according to the composition and application of the synthesized oligonucleotide. Common purification methods are: C18, OPC, PAGE and HPLC.

Quantification: Quantify based on the UV absorption of the oligonucleotide at 260nm.

Storage: Packed and drained.

Q3. Is there any phosphorylation at the 5 'end of the synthesized primer?

The synthesized primer 5 'is a hydroxyl group and has no phosphate group. If you need, you can use the polynucleotide kinase to phosphorylate the 5 'end, or require us to directly phosphorylate at the 5' or 3 'end when synthesizing, which requires additional charges.

Q4. What level of primers are required?

According to experimental needs, determine the purity level of the primers ordered.

application

Primer length requirements

Purity level requirements

General PCR amplification

<60base

HEPD

> 60 base

PAGE

Diagnostic PCR amplification

<40base

HEPD, PAGE

DNA sequencing

Around 20base

HEPD

Subcloning, point mutation, etc.

According to experimental requirements

HEPD, PAGE, HPLC

Gene construction (full gene synthesis)

According to experimental requirements

HEPDPAGE

Antisense nucleic acid

According to experimental requirements

HEPDPAGE

Modified primer

According to experimental requirements

PAGE, HPLC

Q5. How many OD numbers need to be synthesized?

Determined according to the purpose of the experiment. Generally PCR amplification, 2 OD primer, can do 500-1000 times 50ul standard PCR reaction. If you are doing gene splicing or annealing and then connecting, 1 OD is enough.

Q6. How to calculate the primer concentration?

Primers are relatively stable when stored at high concentrations. In general, we recommend that the concentration of primers be formulated as 100 pmol / ul, which is called the preservation concentration, and the working concentration of primers is generally formulated as 10-50 pmol / ul. The volume of added water (microliters) can be directly referred to the recommended volume on the synthesis report, or it can be calculated as follows:

V (microliter) = OD number x 33 x 10000 / molecular weight of primer

The molecular weight of the primer can be obtained from the synthesis report. Note: 1 OD260 = 33 ug / ml.

Before dissolution, you need to check whether the OD numbers of the primers on the synthesis report and the primer labels are the same. If they are inconsistent, please contact us. We can find out the actual output based on the production records.

Q7. How to calculate the Tm value of the primer?

Primer design software can give Tm, which is related to primer length, base composition and ionic strength of the buffer used.

For primers with a length of less than 25base, the formula for calculating Tm is: Tm = 4 ° C (G + C) + 2 ° C (A + T)

For longer oligonucleotides, the formula for calculating Tm is:

Tm = 81.5 + 16.6 x Log10 [Na +] + 0.41 (GC%) – 600 / size ＊

＊ In the formula, Size = primer length.

Q8. How is the molecular weight of the primer (including modification) determined?

The molecular weight (MW) of the unmodified primer can be found on the report sheet provided with the primer. If you need to estimate the molecular weight of a primer, the average molecular weight of each base is 324.5, the molecular weight of the primer = the number of bases x the average molecular weight of the base, or calculate MW = (NA * WA) + (NC * WC) according to the following formula + (NG * WG) + (NT * WT) + (Nmod * Wmod) + (Nx * Wx) + (NI * WI) + 16 * Ns–62.

NA, NG, NC, NT, NI are the number of bases A or G or C or T or I in the primer respectively, WA, WG, WC, WT, WI are the bases A or G or C or T or The molecular weight of I, Nmod, and Wmod are the number and molecular weight of modifying groups, respectively. The molecular weight of the mixed base is the total molecular weight of the mixed base divided by the number of mixing, for example, the molecular weight of the G + A mixture is (313.21 + 329.21) / 2 = 321.21. Ns is the number of thio groups, and each position of thio group increases molecular weight by 16.

Conventional base molecular weight

Base

Molecular Weight

A

313.21

C

289.18

G

329.21

T

304.19

I

314.2

U

290.17

Conventional modification group molecular weight

5'-Biotin

405.45

3'-TAMARA

623.60

5 '-(6 FAM)

537.46

3'-Dabsyl

498.49

5'-HEX

744.13

3 '-(6 FAM)

569.46

5'-TET

675.24

3'-Amino Modifier C3

153.07

5'-Cy5

533.63

3'-Amino Modifier C7

209.18

5'-Cy3

507.59

3'-Thiol Modifier C3

154.12

Q9. How to dissolve primers?

The primer material after drying is very loose. It is best to centrifuge it immediately before opening the cover, or tap the tube vertically on the table to collect the primer powder to the bottom of the tube. Add deionized sterile water or TE (pH 8.0) buffer according to the calculated volume, leave at room temperature for a few minutes, shake to aid, centrifuge to collect the solution to the bottom of the tube. The water used to dissolve the primers is generally not to use distilled water, because some of the pH of distilled water is relatively low (pH 4-5), primers are unstable under such conditions.

Q10. How to save primers?

After the primers are synthesized, after a series of processing and purification steps, they are spin-dried to form a colorless or white flocculent dry powder.

Dry powder: transported at room temperature during transportation, can be stored for one year at -20 ℃.

Storage solution: After preparation, divide into several tubes to avoid repeated freezing and thawing. Store at -20 ℃ for half a year.

Working solution: Normal use, store at 4 ℃ or -20 ℃, but avoid repeated freezing and thawing.

Modified fluorescent primers: need to be protected from light, it is appropriate to use as soon as possible.

Q11. What is wrong with primer inaccuracy?

We occasionally receive reports that users complain about quantitative inaccuracies. The possibility of this situation is:

(1) Quantitative errors, wrong packaging, primer drying or accidental loss during sample collection. This possibility is there, but very few, because as a professional primer synthesis company, our production personnel are strictly professionally trained, this kind of error requires careful avoidance or even elimination. Under normal circumstances, the primers have a sample backup. After receiving the user complaint, we will find out the sample to re-quantify. Generally, there is no problem. If there is indeed a problem, you can arrange to prepare a new one.

(2) Systematic error, we think about 10% is the allowable error. During the use, the working concentration range of the primer is very wide, and a little deviation in the quantitative does not affect the experiment.

(3) When the user receives the primer dry powder, there is no centrifugation or other misoperation before the primer tube cover is opened, which causes the primer dry powder to be partially lost.

(4) The user failed to correctly understand the meaning of the OD number of the primer, and failed to correctly use the spectrophotometer, especially the micro measurement; the user did not correctly convert the OD reading into the OD number in the mother liquor. This situation is more common.

For example, to verify that the amount of standard 2OD primers is accurate, the simple method is: add 1ml of water, dissolve and mix thoroughly, take 100ul, add 900ul of water, use a quartz cuvette with a light path of 1cm, and the wavelength is 260nm. The reading is 0.2.

Q12. How long can the primer be synthesized?

We have synthesized 100base primers, but the yield is very low. Unless necessary, it is recommended that the length of the synthesized fragment should not exceed 80base. The longer the primer, the greater the probability of problems. According to the current primer synthesis efficiency, the percentage of full-length primers that are greater than 80base is not high, and the subsequent processing still loses a lot. The final yield is very low.

Q13. Why is the yield of modified primers lower than ordinary primers and the price is higher?

The main reason is that the modified monomer has poor stability, long coupling time and low efficiency, and the final yield is naturally lower than that of ordinary primers. Modified primers usually require PAGE or HPLC purification, and the purification process has a large loss. The raw material used for the modified primer is several hundred times that of the general primer material, so the price of the product is also high.

Q14. What is the problem that the primer fragments cannot be ligated to the carrier after annealing?

The ligation reaction requires the primer's 5 'phosphate group. If it is necessary to anneal the synthesized primer directly to the corresponding carrier, the primer needs to be phosphorylated. If the phosphorylated product cannot be connected to the carrier, the digestion effect of the carrier needs to be checked, and the primer annealing conditions need to be improved. SiRNA molecules have a special symmetrical structure, and annealing is more difficult. The annealing temperature needs to be increased during annealing.

Q15. If the primer mutation is found by sequencing, is there any compensation? What should I do?

If the sequencing found that the base position of the primer is wrong, we can overlap it for free once without any other compensation or compensation, and do not assume other joint and several liabilities. This is an international standard. The reason we mentioned earlier, the chemical synthesis efficiency cannot reach 100%.

If you encounter this situation, please contact us first, we will check whether the synthetic sequence is consistent with the original order, if you confirm that the primer synthesis sequence is not wrong, we recommend re-picking clones and sequencing, you may find the correct clone. According to our experience, for primers under 40 bases, only 1-2 clones are enough; for more than 40 primers, especially for full-segment splicing, more testing is needed. In general, the location of the mutation in each clone is different. The correct one is always there, that is how to find it. You can also ask us to overlap the primers for free once, but the overlapping primers may contain mutations like the first primers, and will not reduce your chances of encountering problems because of the overlapping primers. In the process of gene splicing, if there are not many mutation points in a section, a few more will be tested, otherwise the primers will be overlapped.

According to the data of the whole gene synthesis, our primer can achieve a fragment of 2000 bases and take three clones, at least one of which is correct.

Q17. Why is the primer OD260 / OD280 less than 1.5?

It should be pointed out that the ratio of OD260 / OD280 cannot be used to measure the purity of the primer. The low OD260 / OD280 ratio is generally due to the relatively high C / T content in the primer. The following table is the OD260 / OD280 ratio of a 20base homopolymer primer, which clearly shows that the OD260 / OD280 ratio is closely related to the base composition of the primer.

A260 / 280 ratios of Crude 20-mer Oligos of Differing Base Compositions

Base Composition

A260 / 280

5-AAAAAAAAAAAAAAAAAAAA-3

2.50

5-GGGGGGGGGGGGGGGGGGGGGG-3

1.85

5-CCCCCCCCCCCCCCCCCCCCCC-3

1.15

5-TTTTTTTTTTTTTTTTTTTTTT-3

1.14

5-AAAAAGGGGGTTTTTCCCCC-3

1.66

Q18. The same OD is detected by PAGE, why the depth of EB staining is different?

The amount of double-stranded DNA (such as plasmid DNA) can usually be judged by EB staining, because EB can be chimeric into double-stranded DNA. Synthetic single-stranded DNA, due to different base compositions, has different possibilities of forming secondary structures, and the degree of EB staining will also be different. For example, Oligo (dT) does not form secondary structures, and EB staining results are very poor. So don't use EB staining method to quantify, but use ultraviolet spectrophotometer to detect.

Q19. How to detect the purity of primers?

The convenient method in the laboratory is to use PAGE method. Electrophoresis was performed using a 16% polyacrylamide gel supplemented with 7M urea. Take 0.2-0.5OD primer, dissolve with urea saturated solution or add dry urea powder to the primer solution until saturated, heat and denature before loading (95 ℃, 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 a voltage of 600V. After a certain period of time (about 2-3 hours), peel off the glue and use a fluorescent TLC plate to detect the band type under the UV lamp. There is no miscellaneous band under the main band, indicating that the purity is good. If conditions permit, it can also be dyed with silver.

Q20. Why the primers that have been dissolved are used normally, but it is not good to use them after a period of time?

If the pH of the water in which you dissolve the primer is too low or contaminates bacteria or nuclease, the primer will degrade. Insufficient thawing and mixing when used, uneven liquid may also cause inaccurate primer addition. It is recommended to separate the primers to avoid repeated freezing and thawing. It is recommended to use 10mM Tris pH7.5 buffer to dissolve the primers, because the pH of some distilled water is relatively low (pH 4-5), the primers are unstable under this condition. There is also a possibility that there is no problem with primers, but the quality of the materials used in PCR, especially the template, is not completely consistent with the previous ones.

Q21. Primers are designed by us, now you can't expand them, should we be responsible?

No responsibility is assumed. We design primers free of charge for customers with insufficient experimental experience and provide some convenience. We follow the general primer design principles after designing the primers and will send you confirmation, and then arrange the synthesis and ensure the quality of the primers. But whether the designed primer can meet your experimental requirements, you must amplify the gene fragments you need, no one can guarantee 100%.

Q22. Is there a problem with the primer quality when PCR amplifies the untargeted fragments?

PCR amplification has no target bands or non-specific amplification. The influencing factors are many and need to be analyzed patiently, such as template structure and quality, reaction conditions, primer design, etc. Various PCR amplification technologies have been developed today, and various high-temperature polymerases are designed to solve the problems of low amplification efficiency and low amplification efficiency encountered in PCR amplification. For example, nested PCR is to amplify gene fragments with very low copy number. Find countermeasures by improving the quality of the template, optimizing the reaction conditions, etc., or redesign the primers if necessary. It is best to set up controls during the experiment to determine the cause. If you doubt the primer problem, please first determine the OD value of your dissolved primer to see if the amount of primer added during the experiment is correct. If the amount is normal, please tell us your primer number, we will review the retained sample. If for unknown reasons, we will re-synthesize it for you for free. If you still cannot expand, please find other reasons. In most cases, we have no problem in rechecking the quality of the primers, because we have strictly controlled the quality before the primers are sold.

Q23. Parameters of commonly used fluorescent dyes

Q24. Is the quality of the primer related to the sequence?

The properties of the four bases and the nature of their respective protecting groups are different. So the difficulty of synthesis is different. The most difficult is the primer with many GC repeats and multiple consecutive Gs in the sequence. Especially for the latter, domestic companies generally cannot make more than 20 G primers. Experiments show that if there are more than three consecutive G structures in the primer, the quality of the product obtained by the traditional method will begin to decline. And the current general desalination, OPC and PAGE methods are invalid.

The HEPD patented technology owned by Dingguo Changsheng Biological Company can overcome the synthesis and purification obstacles of high GC or high G content primers, and can obtain the same very good results for ordinary primers, high GC primers and oligo d (G) regardless of length .