The following primer sets failed to produce an amplicon:
Raw fluorescence data was extracted (No baseline subtraction) and processed with PCR Miner. Data workup/analysis is here. Here is a graph of those primer sets producing an amplicon. All were normalized to actin, which exhibited the smallest amount of deviation across all three samples of the normalizing/housekeeping genes analyzed.
As a preliminary run with these genes, there are a number of promising candidates that could yield some interesting data regarding the physiological response of hard clam to exposure to QPX.
Prepared cDNA using 1ug of RNA from each of the 3 pools (CA, MA, MAX) and processed according to Promega’s M-MLV protocol, using oligo dT primers. Calcs and master mix set up are here. Briefly, RNA was combined with oligo dT primers, denatured @ 70C for 5mins, immediately placed on ice for 2mins, mixed with RT master mix, incubated 1hr @ 42C, 3mins @ 95C, and then stored @ -20C.
Pooled 2ug of each sample in each group (MAX, CA, MA) for a total of 6ug of RNA (3 total samples), brought volume up to 50uL and DNased using Ambion’s Turbo DNA-free following the rigorous protocol. Calcs can be seen here. Spec’d:
All samples look pretty good. Oddly, the 260/280 ratios are absolutely perfect, despite the 260/280 ratios from each individual sample being less than stellar (see yesterday’s EtOH precipiation). Also of note is that the concentrations for all three samples are extremely close, reflecting the accuracy of the NanoDrop readings of each individual sample used for the pool as well as my pipetting.
RNA was stored @ -80C in “Sam’s RNA Box #1.”
Recovered ~50uL from each sample which means each pool yielded ~3.85ug of RNA after DNase treatment. Will proceed with making cDNA from these three pools. In the interest of time (and the failure of our Opticon), I will not verify that these do NOT still contain gDNA (and, it’s pretty unlikely that they do).
RNA was mixed with 0.1 vols of 3M NAOAc (pH = 5.2) and 2.5 vols of 100% EtOH, vortexed and incubated @ -20C for 30mins. RNA was pelleted @ 16,000g, 30mins, 4C. Supe was discarded and pellet was washed with 1mL 70% EtOH. RNA was pelleted @ 16,000g, 15mins, 4C. This was step was repeated a second time. Supe was discarded, the RNA was resuspended in 50uL of 0.1% DEPC-H2O, spec’d and stored @ -80C:
Interestingly, precipitating the samples vastly improved the 260/230 ratios. However, the 260/280 ratios DECREASED for all samples except one (CA 1). Not really thrilled about this fact, nor am I sure why this would happen.
RNA was isolated from the following samples using TriReagent, according to protocol:
MAX 1, 2, & 3
CA 1, 2, & 3
MA 1, 2, & 3
Samples were resuspended in 50uL of 0.1% DEPC-H2O and spec’d:
260/280 ratios are decent for most of the samples, with MAX1 and MAX 2 being the exception. Both of these samples also have very poor 260/230 ratios. Out of curiosity, I will EtOH precipitate all samples to see if I can improve both the 260/280 and 260/230 ratios.
Rec’d package from Scott Lindell @ MBL containing 65 screw cap tubes in a white microtube rack. All tubes are tissues in RNA Later (presumably). One sample (MA4-5) was lost during a brief centrifugation to get tissue sample unstuck from top of tube and in to RNA Later solution. The head of the tube snapped off and the entire tube/sample was obliterated in the rotor. Also, it appears as though all the tubes leaked RNA Later solution during transport. Samples were temporarily stored @ 4C and will be catalogued/transferred to -80C.
Rec’d package of hard clam samples from Emily @ Rutgers on wet ice. Package contained numerous 1.5mL snap cap tubes separated in to groups in zip lock bags. Stored temporarily @ 4C. Will catalog and then store @ -80C.
Rec’d package from Rutgers (Emily Pearson) containing two large Ziplock bags on “wet” ice, each of those containing smaller bags with sample tubes in them. One large bag contains gill tissue samples and the other large bag contains hemolymph samples. Samples will temporarily be stored @ 4C until they can be catalogued and boxed by Lexie later today.
Lexie’s PCR with this primer set and a pool of Mercenria cDNA has yielded contamination in all of her waters. Performed two sets of PCR: one with her existing primer working stocks and the other with a fresh aliquot of primer working stocks. Used my own reagents/water. PCR set up and cycling params are here. PCR ran O/N.
Lane 1 – 100bp ladder
Lane 2 – H2O (Lexie’s primer stocks)
Lane 3 – H2O (Lexie’s primer stocks)
Lane 4 – cDNA (Lexie’s primer stocks)
Lane 5 – H2O (fresh primer stocks)
Lane 6 – H2O (fresh primer stocks)
Lane 7 – cDNA (fresh primer stocks)
All water samples look clean and there’s a nice bright band in the cDNA samples. Lexie’s contamination issue is probably a technique issue and not one of reagent contamination.