Research Oligo

A common method used in laboratories is the PAGE. For electrophoresis, use a polyacrylamide gel with 7 M urea, 20% polyacrylamide gel for primers with less than 12 bases, 16% polyacrylamide gel for primers with 12-60 bases, and 12% polyacrylamide gel for primers with more than 60 bases. Take 0.2-0.5 OD primer, dissolve with urea saturated solution or add urea dry powder into primer solution until saturated, and heat denaturation (95 ℃, 2 min) before loading sample. The purpose of adding urea is to denaturate, and to increase the specific gravity of the sample, which is easy to add the sample. After a certain period of time (about 2-3 hours), strip the polyacrylamide gel and detect the band type with a fluorescent TLC plate under the UV lamp. There is no impurity under the main band, indicating that the purity is good.

(Sometimes due to insufficient denaturation, there may be bands above the main band, which are primer secondary structure bands.)

The OD value of the synthetic primer was determined by using an ultraviolet spectrophotometer with a wavelength of 260 nm and a quartz colorimetric cup with an optical path of 1 cm to determine the optical density of the solution. The optical density of the solution should be diluted to 0.2-0.8. After the DNA dry powder is fully oscillated and dissolved with a certain volume of water, the OD value is measured by diluting with 1 mL of water. OD value of mother liquor should be converted according to dilution ratio. For example, to verify whether the amount of 2 OD primer is accurate, the simple practice is to add 1 mL water, thoroughly dissolve and mix, take 100 μL, add 900 μL water, a quartz colorimetric cup with a light diameter of 1 cm, a wavelength of 260 nm, and the absorbed reading at this time is 0.2.

This situation is more likely to happen when the Oligo DNA is shorter, and the difference between long-stranded Oligo DNA is smaller. There are two main reasons.

(1) different components of A, G, C and T and different electrophoresis speeds.

(2) The steric structure of DNA is different and the electrophoresis speed is different.

The amount of double-stranded DNA (e.g. plasmid DNA) can usually be judged by EB staining, because EB stains nucleic acids by embedding them between the double helix. Synthetic single-stranded DNA, on the other hand, can only be stained by EB by forming a local hairpin loop structure by folding back on itself or by forming a partial double helix structure between the strands. Due to the different base composition, the possibility of forming secondary structure differs from primer to primer, and the degree of EB staining will also vary, for example, Oligo(dT), etc. does not form secondary structure, and the EB staining effect is very poor. Therefore, EB staining cannot be used for quantification, and UV spectrophotometry should be applied for detection.

(1) Primer length generally between 15~30 bases.

(2) Primer GC content between 40% and 60%.

(3) Bases should be randomly distributed.

(4) There should be no complementary sequences in the primers themselves and between the primers

(5) The primers should have relatively high ∆G values at the 5’end and middle ∆G values, and low ∆G values at the 3’ end.

(6) The single strand of the amplification product should not form a secondary structure.

(7) The primers should have specificity.

The longer the primer, the higher the probability of problems. Unless there is a special need, we recommend not to synthesize fragments longer than 80 mer. According to the current primer synthesis efficiency, the percentage of full-length primers will not exceed 40% for a crude product of 80 mer, and a lot will be lost in subsequent processing, so the final yield is very low. The maximum length of primers that can be synthesized is 145 bases.

It is important to use denaturing PAGE electrophoresis for primers. Since the primers are single-stranded DNA, it is easy to form a complex steric structure, so when Agarose electrophoresis is performed, it is easy to have multiple bands or no bands, and it is even more impossible to quantify by Agarose electrophoresis.