All clams appeared to be alive and well. Most had their siphons out when I arrived to start collecting tissues. Clams were shucked after 24hr challenge. Gill and mantle samples were collected in separate 1.5mL snap cap tubes, stored briefly on ice and transferred to -80C in “Hard Clam QPX Challenge 12/2/2009.” box.
Challenged 2 FL hard clams and 1 BX hard clam with ~100uL of unwashed, 11 day old cultures. 2 FL hard clams and 1 BX hard clam received ~100uL of QPX media, as controls. Injections were done through the hinge using 20G1 needles and aimed for the pericardial cavity. After injections, clams were left out of water for 1.5hrs, then return to small containers of sea water. They will be incubated for 24hrs.
Received samples from Scott Lindell today. Two Ziplock bags taped together labelled “11/16/09 Clams scudders.” The bags contain 2mL screw cap tubes with small tissue samples in RNA later. One group of tubes is labelled with FL-3 # and the other group with BX-4 #. Samples will be stored at 4C to be processed later this month.
Received hard clams from Scott Lindell today. Two bags. One group (4 live clams, 1 empty shell) labelled as “FL” and another group (9 live clams) labelled as “BX.” Clams were transferred to separate plastic shoebox containers with sea water and sand. They were stored at 15C until ready for experiment.
Received Hard Clam gill samples on “wet ice” in RNA Later from Rutgers. Samples were collected on 11/4/09 (clams held in refrigerator) and preserved (gill tissue collected) on 11/9/09 according to the paper included with the samples. Samples will be stored @ -80C until we are ready to process.
Submitted four samples (4uL of each) for Agilent Bioanlyzer:
SR01 = total RNA
SR02 = Ambion kit x 1
SR03 = Ambion kit x 2
SR04 = PolyA Tract kit
Precipitation was continued from yesterday. Samples were resuspended in 25uL of The RNA Storage Solution (from PolyAPursit Kit) and spec’d. Samples were stored @ -80C in Sam’s RNA box.
DNased RNA from earlier today was split into four equal parts (175uL = 39.8ug). Three will be used for mRNA isolation and the fourth will remain as total RNA. Three of these were precipitated according to Ambion PolyAPurist Protocol: 1/10 volume 5M ammonium acetate, 1uL glycogen and 2.5 volumes of 100% EtOH. Incubated @ -80C for 30 mins. One sample was processed with the Promega PolyA Tract kit. The remaining two samples were processed according to PolyAPurist Protocol. Of those two, one of the samples was processed a second time to evaluate the effectiveness of running a sample through the PolyAPurist Protocol twice.
mRNA samples were precipitated O/N @ -20C according to the PolyAPurist Protocol.
DNAsed RNA using Ambion Turbo DNA-free kit, following the rigorous procedure. Diluted total RNA to 0.2ug/uL (Vf = 720uL). Added 1uL DNase and incubated the tube @ 37C for 30mins. Added an additional 1uL DNase and continued incubated for 30mins. Added 0.2 volumes of DNase Inactivation Reagent (158.4uL) and incubated at RT for 10mins with periodic mixing. Pelleted inactivation reagent according to protocol and transferred supe (DNA-free RNA) to clean tube.
Results: RNA looks really nice. Have a large quantity of RNA (700uL x 0.2275ug/uL = 159.25ug). Will split into four equal parts and isolate mRNA from 3 of the 4. Those three will be:
Ambion kit x 1
Ambion kit x 2
Promega PolyA Tract kit.
Samples were pelleted and washed with 70% EtOH according to Ambion PolyA Purist protocol. Pellets were resuspended in 10uL of The RNA Storage Solution (included in the Ambion PolyA Purist Kit).
Results: The gill mRNA looks great! Good yield and good ratios. Hemocyte mRNA looks kinda rough and a very low yield (which was to be expected).