When measuring OD values, the displayed values on the spectrophotometer represent the OD per milliliter of the solution.

If the OD value is measured as 1.5 for a 200 ml solution, the overall OD for the solution would be calculated as follows :

Overall OD=OD value×Volume in liters

Overall OD=1.5 OD/ml×0.2 L=0.3 OD

So, in this case, the overall OD for the 200 ml solution would be 0.3 OD.

The Oligo DNA in the solution should be fine at room temperature for a few days (3~4 days), but it is advisable not to store it for more than a week.

Its shelf life is influenced by factors such as bacteria, nucleases, and other components present in the solution.

Whether it is PAGE purification or HPLC purification, increasing the purification quantity significantly decreases the purity of the product. Therefore, to ensure product quality, we generally control the purification quantity to around 2 OD.

Providing a large quantity with a high OD for the sake of purity is impractical and unscientific.

Typically, a 20-mer DNA product with 2 OD (Approximately 10 nmol) can be used for around 400~500 PCR reactions (Total volume 50 µl, Primer concentration = 10 µM), as well as 3000 sequencing reactions (Primer concentration = 3.2 µM).

Therefore, a 2 OD quantity of DNA is sufficient for most experimental work.

Those who have conducted Oligo DNA PAGE electrophoresis are aware that the brightness (or clarity) of bands for DNA products of different sequences but with the same OD varies. This is due to the staining with dyes like EtBr, which permeate between the double strands of DNA, whereas synthesized Oligo DNA is single-stranded. The more complex the three-dimensional structure formed by the Oligo DNA itself, the easier the staining with EtBr, resulting in brighter bands.

Conversely, some Oligo DNA, due to the lack of three-dimensional structures, may not be stained by EtBr at all.

Therefore, we believe that it is unrealistic for some companies to provide electrophoresis images with similar brightness for all products.

It is essential to use denaturing PAGE electrophoresis for Oligo DNA.

Oligo DNA is single-stranded, and it readily forms complex three-dimensional structures.

Therefore, when subjected to Agarose gel electrophoresis, multiple bands may appear. (Unable to conduct quantitative analysis using agarose gel electrophoresis.)

We believe that the main reasons for this phenomenon are as follows :

Differences in the composition of A, G, C, and T, leading to variations in electrophoretic mobility.

Variations in the three-dimensional structure of DNA, resulting in different electrophoretic mobilities.

This situation is more likely to occur with shorter Oligo DNA sequences, while the differences between long-chain Oligo DNA are comparatively smaller.

Most manufacturers attribute the occurrence of such issues to :

1. Mismatches during the PCR process.

2. Mutations during the cloning process.

While we cannot deny these possibilities, they are considered highly unlikely. After a thorough analysis of this situation, we have identified some potential reasons for reference :

1. Sudden blockage in the synthetic machine pipeline can lead to an instantaneous interruption of chemical reactions, potentially resulting in the loss of individual (or a portion of) nucleotides.

2. Malfunction in the control program, leading to errors in synthesis.

3. Coupling between bases before attachment to the DNA fragment during the synthesis process, followed by attachment to the DNA fragment, resulting in multiple-base phenomena.

4. Conversion of the G base to its enol isomer during synthesis, causing G to transform into A during PCR reactions.

5. Depurination during synthesis, where the bases after depurination are not recognized during DNA polymerization, leading to observed losses of A or G. The higher the purine content, the more likely this situation occurs.

6. Incomplete deprotection results, with C being deprotected faster than G. Incomplete deprotection can lead to the loss of bases.

7. Incomplete capping reactions during synthesis, allowing short DNA fragments to continue participating in synthesis, causing base losses.

It should be noted that the likelihood of these scenarios is extremely low. However, as the length of the synthesized DNA chain increases, the probability of such events also increases.

★Please confirm :

1. Due to the impossibility of 100% primer purity, it is possible that impure primers were selected during cloning, resulting in the cloning of PCR products amplified with contaminated primers. In this case, please consider reselecting a clone for sequencing; the results may improve.

2. If sequencing results do not improve after selecting 2~3 clones for sequencing, we will re-synthesize the primers free of charge.

3. Are the reagents for the PCR reaction working properly ?

4. Is the PCR instrument functioning correctly ?

5. Are the PCR reaction conditions appropriate ?

★If a protein expression experiment has been unsuccessful for several months and sequencing reveals errors in the primers, what to do :

1. Before conducting a protein expression experiment, it is essential to validate the DNA sequence, which is common practice in experimental procedures.

2. We can re-synthesize the primers free of charge.

3. In the case of a claim, according to international industry practices, the scope of the claim is limited to the product's price range.

We can provide synthesized primers with a length ranging from 6 to 75 nucleotides.

When the primer sequence is longer, it becomes challenging to guarantee the accuracy of every sequence in the product due to limitations in synthesis and purification methods.

Since the 5' end of primers commonly used for PCR does not have a phosphate (P), the resulting PCR product also lacks a phosphate at the 5' end. When cloning into a dephosphorylated blunt-ended vector, cloning is not successful.

On the other hand, cloning into a non-dephosphorylated blunt-ended vector leads to a high background. In such cases, please perform phosphorylation (P) treatment on the 5' end of the PCR product.

The reasons for PCR failure can be considered from several aspects :

1. Are the primers and template well-matched, and what is the level of homology ?

2. Do the primers have three-dimensional structures, or is there a higher-order structure between the two primers ?

3. Are the reagents for the PCR reaction working properly ?

4. Is the PCR instrument functioning correctly ?

5. Are the PCR reaction conditions appropriate ?

If everything appears normal and the problem persists, we can re-synthesize the primers free of charge. If the issue persists even after re-synthesizing the primers, please send both the primers and the template to our company, and we can assist in troubleshooting the PCR reaction conditions.

Currently, we do not offer this service.

We only provide general primer synthesis.