Tag Archives: hemolymph

RNA – Precipitation continued from yesterday

Transferred supe to a fresh tube and added 1mL 70% EtOH to remaining pellet. Spun samples max speed @ 4C 30 mins. Removed supe and washed pellets with 1mL 70% EtOH. Spun max speed 10 mins. Removed supe . Resuspended the “supe” sample in 50uL 0.1%DEPC-H2O and the “pellet” sample in 100uL 0.1%DEPC-H2O.

Results: 260/280 ratios look good. The 260/230 ratios are still horrible. Total yield from these two samples are ~5ug. Will get more hemolymph from clams in order to use more total RNA in the mRNA isolation to maximize cost saving.

RNA – Reprecipitation of hard clam RNA from yesterday

Because of the relatively large size of the pellets vs. the amount of RNA, I think another round of precipitation would be best to help remove additional residual salt carryover. Will precipitate O/N according to Ambion PolyA Purist protocol. RNA pellets were resuspended in 250uL of 0.1%DEPC-H2O and precipitated O/N @ -20C.

NOTE: Upon adding 100% EtOH to sample, the solution turned very cloudy and a white precipitate immediately formed inside the tube. I do not think this precipitate is RNA. Tomorrow, before spinning the tube, I will transfer the supe to a fresh tube and process both tubes simultaneously. Hopefully this will remove/eliminate most of the excess salt or whatever seems to be forming the pellet.

Bleeding – Hard Clams

Bled 8 clams from 20090108 and 20090109, #4, 6, 8, 15, 16, 17, 21, 26. Bled clams using a 23g 1.5 needle on a 3mL syringe. Fluid was gathered and ranged from ~0.4-1.0mL. Hemolymph was transferred to individual 1.5mL snap cap tubes and spun @ 100g for 30mins @ 4C. Most of the supe was removed, but left ~100uL in each tube to avoid disturbing any pellet. Samples were stored @ -80C in the red box with previous hard clam hemo samples.

Pellets were apparent in all 8 samples, whereas they had not been noticeable before in last week’s bleeds.

Also, 3 clams were found with cracked shells, but alive, including the one pictured below that is split open entirely.

RNA Isolation – Hard Clam hemolymph from 20090108, 20090109

1mL of TriReagent was used to isolate RNA from 3 combined tubes of hemolymph. This resulted in 10 total RNA preps. Pellets were resuspended in 100uL of 0.1% DEPC-H2O and pooled into a single tube and NanoDropped.

Results: RNA solution looked very cloudy and contains a fair amount of insoluble “stuff”. 260/280 ratios also looked bad. Will precipitate O/N according to Ambion PolyA Purist Kit before isolating mRNA tomorrow.

Bleeding – Hard Clams

Bled 24 hard clams using a 23g 1.5 needle on a 3mL syringe. Fluid was gathered and ranged from ~0.4-1.0mL. Hemolymph was transferred to individual 1.5mL snap cap tubes and spun @ 100g for 30mins @ 4C. Most of the supe was removed, but left ~100uL in each tube to avoid disturbing any pellet. Samples were stored @ -80C in the red box wiht previous hard clam hemo samples. Clams were numbered and transferred to a holding tank. Gill and mantle tissue was collected from 10 of the clams. The collected tissue and the rest of the carcasses were stored @ -80C.

Bleeding – Hard Clams

Bled 6 hard clams using a 23g 1.5 needle on a 3mL syringe. Fluid was gathered and ranged from ~0.4-1.0mL. Hemolymph was transferred to individual 1.5mL snap cap tubes and spun @ 100g for 30mins @ 4C. Most of the supe was removed, but left ~100uL in each tube to avoid disturbing any pellet. Samples were stored @ -80C in the red box with previous hard clam hemo samples. Clams were numbered and transferred to a holding tank.

SDS/PAGE/Western – anti-HSP70 Ab Re-test

Another attempt to determine appropriate amounts of anti-HSP70 Ab (ABR cat# MA3-006)and/or protein needed for better detection of HSP70 in Gigas protein samples. Gigas gill protein extracts from 20080617 (both control and Vibrio exposure samples) were each pooled. The two samples were mixed with an equal volume of 2x sample reducing buffer. 100uL of hemolymph were extracted from Gigas muscles and mixed with an equal volume of 2x sample reducing buffer. Samples were boiled for 5mins. and loaded onto a Pierce 4-20% tris-hepes gel. Also loaded 10uL of SeeBlue ladder. Ran gel @ 150V for 45mins. Samples were transferred to nitrocellulose 20V for 30mins. Well locations were marked on the membrane with a pencil. Gel was stained with Coomassie stain for 30 mins and then destained with 10% acetic acid until bands were clearly visible.

external image 20081216.JPG

Lane 1 – Ladder
Lane 2 – Control (20ug)
Lane 3 – VE (20ug)
Lane 4 – Hemos (40uL)
Lane 5 – Control (10ug)
Lane 6 – VE (10ug)
Lane 7 – Hemos (20uL)

Lane 8 – Control (20ug)
Lane 9 – VE (20ug)
Lane 10 – Hemos (40uL)
Lane 11 – Control (10ug)
Lane 12 – VE (10ug)

Membrane was cut in two (between lanes 7 & 8) for probing with two different primary Ab concentrations: 1:5000 (per the manufacturer’s suggestion) and 1:2500. Membranes were incubated 1hr. in 15 mL of blocking solution. Primary Ab was added at the two above mentioned concentrations to the respective membrane and incubated 1hr. Membranes were washed with 1x TBS-T 3 x 10mins. Secondary Ab (DAM-HRP) was added with blocking solution to the membranes at a 1:2500 dilution and incubated 30mins. Membranes were washed with 1x TBS-T 3 x 10mins. Membranes were developed with Millipore Immobilon Western Chemiluminescent reagent and imaged together.

Results:

No image of any sort! Not even the pencil marks were visible. Just a blank screen. I’m starting to suspect that something is wrong with the imaging system or something. This is basically a repeat of the Western on 20081210 which worked. Now I’m not sure what to do at all. This blows.