DNA Extraction with Prep and Plasmid PCR progress

 Introduction: 

This week included three major advancements in two projects. The majority of the week went towards efforts in sequencing, in DNA extraction of D. sonorensis and then DNA sampling prep for Illumina NEBNext sequencing for both the D. sonorensis and the D. caeni sample extracted the previous week. Both preps were attempted to be sequenced, with one run of D. caeni successfully completed with a genome coverage of 99.999% and the run of D. sonorensis unable to be completed due to technical issues. The latter part of the week was dedicated to trying to identify the problem in the long amp PCR protocol of the 7kb plasmid from D. aquaticus , which is set to be amplified with Gibson primers to attempt Gibson assembly with the already attained Cmr gene. The week ended in mixed results, but with directions for future attempts. 

Methods: 

D. sonorensis DNA extraction was completed with the DNeasy QUIAGEN kit, with slight alterations to the original protocol. Cells were grown on plate for approximately 48 hours and gram stained to verify lack of contamination and cell identity. Plates had been inoculated in a lawn to provide significant cell density for transferring to 0.7 mL of TGY broth and ground with a micro-mortar two times, about five minutes each time. After each round of mortaring, cells were vortexed at max speed for thirty seconds. It was observed after two rounds that plaque colonies were still visibly present but had been reduced in size enough to allow for acceptable pipetting without issue of sticking to the inside of the tip, which had been described previously. This allowed for the transfer of all collected plaques into the DNeasy protocol, in which bead beating was completed for a total of 10 minutes at max speed (5m/s) one minute on and one minute off with incubation on ice. Sample was eluted in 25 ul to retain high concentration, with an added 5 minute incubation at room temperature prior to centrifugation. Sample was stored in the minus 80 for future use in DNA Illumina prep protocol. 

DNA prep for sequencing was completed for the D. caeni and D. sonorensis samples using the standard single sample protocol from NEBNext for purification with purifying beads. Each sample was diluted to an initial input of 85 ng, so that a 63.7 ng/ul  (260/280-1.90, 260/230-2.04) sample of D. sonorensis had an input of 1.30 ul. D. caeni sample diluted to 133.0 ng/ul (260/280-1.93, 260/230-2.01) had an input of 0.65 ul. The D. caeni sample was loaded the same afternoon post sample purification with a 1 ul total input  elution diluted with 0.1X TE buffer solution. D. sonorensis final elution was further diluted to a 1:100 (D. caeni was diluted only 1:20 in comparison) and 20 ul of this solution was loaded into a separate flow cell the following day. 

The amplification of the 7kb in its entirety and the addition of Gibson hangovers to prepare for plasmid engineering was attempted again, doing two separate experiments. The first included the same master mix and PCR conditions from the first attempt, using a 50 sec/kb extension time and an annealing temp of 59 degrees Celsius. Two samples, one the M23 gene as a positive control and the other the 7kb primers,  were from "colony" PCR conditions of a 5 minute boil prior to PCR. The second pair of identical primers were using plasmid extraction template. The only results visible on the post PCR gel was the M23 gene from the colony template. A heat gradient was carried out from 50 to 66 degrees and an extended denaturation time was used with colony template. PCR products were run on a 1% agarose gel. 

Results: 

The intial D. sonorensis DNA extraction produced a 697.6 ng/ul sample with a 260/280 of 1.91 and 260/230 of 2.11. When visualized on an agarose gel, the approximate concentration was confirmed as the fluorometer was not functional at this time. 

The first sequencing run with D. caeni was successful, producing raw data that has undergone preliminary bioinformatics processing. 

The D, sonorensis sequencing attempt was initially running, but stopped at some point for an unknown reason. It was determined to be a technical difficulty with the flow cell as opposed to sample or user error, and this sequence will be attempted again with the same final sample elution achieved from this week. 

The 7kb PCR amplification was also ultimately unsuccessful, with no bands being visibly present on the post heat gradient agarose gel. 

Discussion:

This week was wildly successful for one species in particular, D. caeni, in which what appears to be a very usable and productive sequence has been achieved. The future will include a long fragment sequence run in order to resolve the replicon structure and and finalize a hybrid assembly of sequences. There will undoubtedly be plenty of future questions and discussion regarding the data revealed in this sequence run. Unfortunately the same cannot be said for D. sonorensis, which has proven to be very characteristic of the species. Since it was initially verified from tech support that it seems to be a flow cell issue, another run will be carried out next week with a slight adjustment to the final concentration by decreasing the level of dilution from 1:100 to about 1:10 or 1:20 per the suggestion of the company. We can only hope this adjustment and another functional flow cell with finally reveal the long sought after sequence of D. sonorensis shortly. Finally, the 7kb will still need to be adjusted for success. It has been debated that additional supplements may be needed for a successful amplification, but it is still very unclear what the exact next steps will be for what can be altered since absolutely no amplification seems to be taking place in any significant amount. It seems very odd that the template of plasmid was unsuccessful when logically it should have worked more so than the colony sample. It may need to be reevaluated from the beginning steps to ensure the reaction is being carried out with the best chances of success, including the primers or verifying the plasmid extraction template as being still good.