Reverse transcription was performed using 100ng of each sample with M-MMLV Reverse Transcriptase from Promega.
Briefly, 100ng of DNased RNA was combined with oligo dT primers and brought up to a final volume of 15uL. Tubes were incubated for 5mins at 70oC in a PTC-200 thermal cycler (MJ Research), using a heated lid. Samples were immediately placed on ice.
A master mix of buffer, dNTPs, water, and M-MMLV reverse transcriptase was made, 10uL of the master mix was added to each sample, and mixed via finger flicking. Samples were incubated for 1hr at 42oC in a PTC-200 thermal cycler (MJ Research), using a heated lid, followed by a 5min incubation at 65oC.
Samples were stored on ice for use later this afternoon by Ronit.
Used 1000ng of RNA in a 50uL reaction in a 0.5mL thin-walled snap cap tube. Samples were mixed by finger flicking and then incubated 30mins @ 37oC in a PTC-200 thermal cylcer (MJ Research), without a heated lid.
DNase inactivation was performed (0.1 volumes of inactivation reagent; 5uL), pelleted, and supe transferred to new 1.7mL snap cap tube.
Samples were stored on ice in preparation for qPCR to test for residual gDNA.
Continuing Illumina’s generous efforts to use our geoduck samples to test out the robustness of their emerging sequencing technologies, they have requested we send them some geoduck tissue so that they can try to complete the genome sequencing efforts using the 10x genomics sequencing platform.
Isolated additional gDNA for the genome sequencing. In an attempt to obtain better yields, I used ctenidia (instead of adductor muscle). Additionally, to try to improve the quality (260/280 & 260/230 ratios) of the gDNA, I added a chloroform step after the initial tissue homogenization.
Yield is definitely much, much better than adductor muscle! Should’ve switched to a different tissue a long time ago! We should finally have sufficient quantities of gDNA to allow for BGI to proceed with the rest of the genome sequencing! Will run sample on gel to evaluate integrity and then send off to BGI.
The NanoDrop & Qubit numbers still aren’t close (as expected).
The addition of the chloroform step definitely helped improve the 260/280 OD ratio (see below). However, the addition of that step had no noticeable impact on the 260/230 OD ratios, which is a bit disappointing.
The remaining Olympia oysters from Jake Heare’s reciprocal transplant experiment have been retrieved from field sites and are awaiting sampling. The oysters have been stored in the cold room (temp?) for 15 days so far.
The remaining Olympia oysters from Jake Heare’s reciprocal transplant experiment have been retrieved from field sites and are awaiting sampling. The oysters have been stored in the cold room (temp?) for 6 days so far.
Sampling scheme is as follows:
Assign unique number to oysters
Photograph with ruler for future shell measurements
Dissect ctenidia for DNA isolation
Dissect & discard viscera (e.g. digestive gland and gonad)
Performed reverse transcription on the Olympia oyster DNased RNA from the 1hr post-mechanical stress samples from Jake’s project. To accommodate the large numbers of anticipated genes to be targeted in subsequent qPCRs, I prepared 100μL reactions (normally, 25μL reactions are prepared) using 250ng of each DNased RNA. A 1:10 dilution of the oligo dT primers (Promega) was prepared to improve pipetting accuracy. All incubations were performed in a thermal cycler without using a heated lid.
DNased RNA was combined with NanoPure H2O and oligo dT primers in 24 wells of a PCR plate, heated @ 70C for 10mins and immediately placed on ice. After 5mins, the plate was spun 2000g @ RT for 2mins and returned to ice.
25.25μL of a master mix containing 5x M-MLV Buffer (Promega), dNTPs (10mM each; Promega), and M-MLV Reverse Transcriptase (50U/rxn; Promega) was distributed to each well and mixed via pipetting. The plate was heated @ 42C for 1hr, 95C for 3mins. The plate was spun 2000g @ RT for 2mins and then stored @ -20C.