High Pressure Liquid Chromatography (HPLC) is a separation method that can be used to separate oligonucleotides by their sequence and is sensitive enough to give single nucleotide resolution. In other words, HPLC can differentiate between two nearly identical oligonucleotide sequences. This property makes this analytical method very useful for characterizing the products from the degradation of a longer oligonucleotide into smaller chains.
Matrix-Assisted Laser Desorption/Ionization Time Of Flight (MALDI-TOF) is a mass spectrometry technique that is extensively used for the analysis of biomolecules, and has been employed as a valuable means for determining DNA sequence. We used this as a means for determining the sequences of the degradation products. This information, in turn, tells us which linkages along the chain are most prone to degradation, allowing for the potential of engineering a new sequence that will degrade into predetermined products.
The nanodrop uses the degree to which a substance absorbs a certain wavelength of light to provide fast and reliable measurement of the concentration of a given substance. This was an invaluable technique for ensuring that our oligonucleotide solutions were all at the same concentration.
First of one of the two methods used to incubate the experimental samples. Incubation in this manner allows the samples to be monitored more easily while also incubating each sample tube individually.
The second method used for sample incubation. The convection oven was kept at an identical temperature to the block incubator. It differs from the block incubator by changing the evaporation of the oligonucleotide buffer (Deionized Water) so that it does not condense at the top of the tube and instead maintains it primarily in a vapor phase. Additionally, the oven was used to dry samples that were deposited on either a glass or polypropylene surface.
A dehydration method similar to lyophilization that increases the temperature of the environment in order to speed up evaporation processes. This machine was used to dry some of our experimental samples prior to incubation.
This is a separation column that is a simple and easy-to-use method for swapping buffers for oligonucleotides via gravity flow. For this investigation, Nap-5 columns were used to exchange buffers after HPLC purification so that the samples could be later analyzed with MALDI-TOF.