Great news! No amplification in the 08:4-1 (positive for WS, but naive for phage)!! These results strongly suggest that these primers are specific for the WS bacteriophage! This is really cool and exciting. Next steps will be to confirm via in-situ hybridization (ISH).
Additional summary of the results:
Primer set ORF25_CSF shows the highest sensitivity.
Primer set ORF20_CSF fails to amplify anything in 06:6-53
Quantified gDNA from the following samples in preparation for high-throughput sequencing by Stan Langevin’s group. Quantification was done using Pico Green, Tecan plate reader and Magellan 6 software. The r^2 value of the standard curve was 0.9986 and replicates showed little variation.
Concentrations below are the mean of three replicates and are in ng/uL.
06:5-28 – 42.4
06:6-41 – 60.3
06:6-44 – 57.1
06:6-53 – 72.1
06:6-54 – 40.1
06:6-55 – 40.5
06:6-66 – 40.6
Stan Langevin has requested at least 50ng of DNA from each sample. I will aliquot ~100ng of each sample into individual tubes. He will be picking up aliquots tomorrow morning for sequencing on the MiSeq (Illumina).
Isolated RNA from 3 digestive gland (DG) samples: 08:13-7, 08:13-9, and 08:13-16 using the RNA Power Soil Kit (MoBio) according to the manufacturer’s protocol and spec’d on the Roberts Lab NanoDrop1000.
Isolated RNA from Red Abalone Dg (10:25-24, 25, 26) using TriReagent, according to protocol. These samples are expected to have high levels of the Rickettsia phage that I’ve been attempting to investigate. The tissue samples were taken on 20110224 by Lisa and stored in RNA Later @ 4C. Remaining tissue will be returned to existing tubes and stored @ -80C. Previously, RNA from Ab Dg had been isolated using the MoBio RNA PowerSoil Kit due to its ability to produce extremely clean RNA from Dg (a nasty tissue). However, we currently do not have that kit. RNA was resuspended in 100uL of 0.1% DEPC-H2O and spec’d on the Roberts Lab ND1000. RNA will be stored in “Sam’s -80C Box #1″ in the Roberts’ Lab -80C freezer.
As expected, the RNA quality is low (based on OD260/280), as the TriReagent method of isolation does not clean up the RNA as extensively as the MoBio RNA PowerSoil Kit which has been previously used. Additionally, the RNA solution is brown colored. Will DNase RNA.
DNase – Abalone Dg RNA (from earlier today)
DNased Abalone Dg RNA that was isolated earlier today using Ambion’s Turbo DNA-free Kit following the “rigorous” protocol. Briefly, 5ug of RNA was brought up to 45uL with nuclease-free H2O. Reaction was brought to 50uL with 10x DNase Buffer and 1uL of DNase. Sample was incubated @ 37C for 30mins. An additional 1uL of DNase was added, mixed and incubated for 30mins. DNase was inactivated, supe transferred to clean tube and spec’d on Roberts’ Lab ND1000. DNased RNA will be stored in “Sam’s RNA Box #1″ in the Roberts’ Lab -80C freezer.
OD260/280 values still look poor (which was expected based on initial isolation from earlier today). Will check DNased RNA for residual gDNA carryover.