Primer/Probe What is it?
Primers (Oligonucleotides) are short sequences of nucleotides. They are used in many molecular and genetic procedures. Nucleic acid probes are very important reagents for detecting specific nucleic acid sequences by hybridization. Visualization of a specific hybridized probe in solution or added to the solid phase is the basic principle of the hybridization assay.
The specificity of the primers and probes to the oligonucleotide sequence should be checked by quality control tests. Standard desalted (DSLT) purification and HPCL purification options can be offered as primer purification processes. Whichever purification method is preferred, the consumption will vary according to the final concentration used in the PCR process. Therefore, the estimated number of PCR reactions given according to the synthesis scale prepared varies considerably.
Lyophilized primer/probelar can be stored at room temperature as it is not affected by temperature changes. After reconstitution, it should be stored at -20 ºC. For long-term use, it should not be freeze-thawed and should be aliquoted.
Primer/Probe How to Reconstitute?
TE (Tris-EDTA buffer solution) or DNase/RNase free water (DNase/RNase free water/DEPC water) is used to solubilize oligonucleotides.
- 100 μM = 100 μMoles/L = 0.1 nMoles/μL
- Master stock = 100 μM
Amount of Oligo
34.0 = 155.5 = 0.96
OD 260 nMoles mg
- 100 μM = X nmol lyophilized primer + (X × 10 μl H2O)
- For example , if 155.5 nmol of primer is present, 1555 μl H2O is added to create a 100 μM primer master stock.
- If working stock = 10 μM
- Dilute the primer master stock with H2O 1:10 in a sterile microcentrifuge tube.