The aim of this procedure is to successfully amplify hamster adapted PreOn strains in vitro. This is accomplished by first homogenizing uninfected hamster brain to be used as a substrate, and then preparing the infected tissue to be used as a seed. The second step is to mix the substrate and the seed in PCR tubes, and then perform protein misfolding, cyclic amplification or PMCA by sonication and incubation cycles.
Next, the amplified material is diluted with fresh substrate and serially amplified in vitro, and then perform PMCA. Finally, the samples are digested with proteinase K and then analyzed by western blot. Ultimately, the increase in band intensity in the western blot can be used to determine the in vitro amplification of the prion strain.
This method can help answer key questions to the PreOn field, such as what are the important co-factors involved in PreOn amplification? Generally, individuals new to this method will struggle with little but important details involving the technique, as well as to avoid sample cross-contamination. Before homogenizing wipe the pipettes with 1%bleach, saturate the sonicate PCR tube rack tissue homogenizers and PCR tube storage racks with bleach as well.
After 10 minutes, rinse each piece of equipment thoroughly with distilled water and then cover the work area with a fresh Verly dry lab Soaker. Now use a 10 buric tissue grinder and freshly prepared ice cold conversion buffer to homogenize an uninfected hamster brain to a 10%weight per volume solution. For uses the PMCA substrate solution, then place the homogenate in 15 milliliter to conical tubes and clarify the homogenized solution by centrifugation at 500 times G for 30 seconds.
Next, being careful to avoid the large pellet aliquot, the resulting snat into 1.5 milliliter micro centri tubes. To prepare the PMCA seed solution, homogenize harvested infectious material to a 10%weight per volume solution as just demonstrated, but with daros phosphate buffered saline or DPBS instead of conversion buffer, and then eloquent the solution into 0.6 milliliters of micro centrifuge tubes. Before beginning the amplification procedure, cover the work area with a fresh, firstly dry lab soaker at the PMCA substrate to a 200 microliter thin walled PCR tube strip and dilute the PMCA seed into it at a ratio of one to 20, place a total of four replicates in red labeled tubes.
Place two replicates of a 15 microliter aliquot of each sample in blue labeled PCR tubes for use as a minus 80 degrees Celsius unsated control. Place an additional two replicates of a 15 microliter aliquot from each sample into PCR tubes to be stored at 37 degrees Celsius as an additional unsated control. Then place the minus 80 degrees Celsius unsated controls in a minus 80 degrees Celsius freezer and place the other unsated controls in a 37 degrees Celsius incubator for the duration of the PMCA reaction.
Now begin the first PMCA amplification round by placing the PCR tube strip containing the remaining 70 microliters of each sample into a sonicate connected to a recirculating water bath set to a constant temperature of 37 degrees Celsius. A PCR tube containing PMCA substrate alone can be included as a mock control to check for cross-contamination nation fill no more than 30%of the sonata's tube rec by spreading the PCR tubes throughout the microplate horn. Keep at least one row of empty space between the PCR tube strips and allow no more than four tubes to be connected together per strip.
The amplification of P-R-P-S-C varies throughout the microplate horn. The best P-R-P-S-C amplification efficiency is obtained within a radius of five centimeters from the center of the microplate horn. One round of PMCA consists of 144 cycles with five seconds of sonication and 10 minutes of incubation per cycle for about 24 hours per round of PMCA while fornicating adjust the amplitude to have a power output ranging between 155 to 170 watts.
Place an aquarium heating pad on the sonicate lid to avoid water condensation, which could lead to sample cross-contamination. Shake the tubes every five hours to avoid water condensation on the domed caps of the PCR tubes after the round has been completed. Remove the tube strip from the ator and spin down the samples.
Then gently vortex the tubes, keeping the solution from the cap to avoid contamination when the caps are opened to further minimize contamination when opening the tubes. Avoid touching the rim and place the cap on a clean Kim wipe. Then for serial PMCA of the PreOn strain transfer five microliters of the sonicated sample into 95 microliters of fresh PMCA substrate.
Use new gloves for each sample. Next, remove two 15 microliter aliquots from each of the sonicated samples and store one aliquot minus 80 degrees Celsius and the other at 37 degrees Celsius. For the unsated control solutions for PMCA round two subject the remnants to a second PMCA round as just demonstrated.
This step can be repeated as many times as desired. A PCR tube containing PMCA substrate of the uninfected brain alone can be included to check for cross contamination after syndicating the samples for the desired number of rounds, mix five microliters of 18 micrograms per milliliter of proteinase K with five microliters of the sonicated sample, the minus 80 degrees Celsius control and the 37 degrees Celsius. Control in separate PCR tubes and digest the sample at 37 degrees Celsius for one hour.
Then add 10 microliters of gel loading buffer to the digested sample and boil the solution for 10 minutes. After it calls, load 10 microliters of the sample gel loading buffer mixture onto a Western block gel. To determine the amount of amplified P-R-P-S-C, compare the amount of proteinase k digested sample in the Western blot to either the unsated controls or to known amounts of proteinase.
K undigested recombinant. P-R-P-C-P-M-C-A is used to amplify P-R-P-S-C in vitro successful amplification of the hamster adapted PreOn protein hamster CWD and drowsy TME can be seen in this western BLO analysis by comparing the band intensities before and after PMCA proteinase K digestion of P-R-P-S-C followed by Western BLO analysis shows an upper, middle and lower band corresponding to the dai mono and glycosylated prion protein respectively. Some PreOn strains can be differentiated by their electrophoretic mobility.
For instance, there is a two kilodalton difference in migration between the hamster CWD and drowsy TME PreOn strains. This high fidelity amplification is observed by similar electrophoretic migration of the PMCA amplified PreOn hamster CWD strain compared to its corresponding hamster CWD seed as well as for the drowsy TME strain as compared to its drowsy TME seed. If no cross contamination or de novo, P-R-P-S-C formation occurs Western blot analysis, OFM control PMCA samples should remain clear after proteinase k digestion.
While attempting to do this procedure, it is important to remember to clean every piece of equipment before using a new sample, thus avoiding cross contamination.
