To this day, the Q-FISH method continues to be utilized in the field of telomere research. Q-FISH utilizes this unique characteristic of PNAs where at low ionic strengths, PNAs can anneal to complementary single-stranded How to determine hybridization sp sp2 sp3 pdf sequences while single-stranded DNA cannot. Q-FISH is able to quantify the hybridization of PNAs to telomeric sequences.
This will swell the collected cells and spread the chromosomes. Place a few drops of the cell suspension onto a microscope slide and let air dry overnight. Wash slides several times with PBS. A small volume of the hybridization mixture is placed onto a coverslip and then placed gently onto the microscope slide which contains the fixed cells. The slide is then left at room temperature for several hours to allow the PNA to hybridize to complimentary DNA.
Slides are then carefully washed in various wash solutions to remove unbound PNA. Microscope mounting medium is then placed onto the cells. Before experimental samples are imaged, fluorescent reference beads are imaged in order to ensure the proper set-up of the camera and fluorescent microscope. In addition, these reference beads will be imaged prior to every acquisition session. This will ensure that the differences between samples are not due to errors in the lamp or camera.