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.

PCR – Dungan Isolates

Samples (in Chelex) were vortexed and heated @ 95C for 30mins with periodic vortexing. Tubes were spun max speed @ 4C for 2 mins to pellet Chelex. Set up PCR using Immomix master mix. Anealing temp. = 56C. PCR set up here.

Lane 1 – 100bp ladder

Lane 2 – xCvC-11t

Lane 3 – xCvC-12t

Lane 4 – xCvC-17t

Lane 5 – VNTc-12-C1/G10

Lane 6 – BC05Ca-18t/H5

Lane 7 – VATm-1.2t

Lane 8 – VNTc-1.5t

Lane 9 – Neg. Control

Results: PCR seems to have worked for some of the samples. The bottom-most band in lanes 4, 5, 6, 9, & 10 were cut out and stored in “Sam’s Misc. -20C Box”. Date is 1/8/2009, since this PCR ran O/N.

PCR – Dungan Isolates

All samples , except xCVC-11t, are already in Chelex. For xCvC-11t, pipetted a shunk of cells/tissue from source tube. Volume of liquid (EtOH) was ~75uL. Added this to screw cap tube containing 300uL of 10% Chelex solution. Vortexed and incubated @ 95C for 30mins. Vortexed and incubated other samples at 95C for 5mins. Set up PCR with AmpliTaq. Anneal temp. = 53C. PCR set up here.

Lane 1 – 100bp ladder

Lane 2 – xCvC-11t

Lane 3 – xCvC-12t

Lane 4 – xCvC-17t

Lane 5 – VNTc-12-C1/G10

Lane 6 – BC05Ca-18t/H5

Lane 7 – VATm-1.2t

Lane 8 – VNTc-1.5t

Lane 9 – Neg. Control

Results: Ladder is degraded and there are no bands in any lane. Will repeat and try to duplicate Steven’s results from 20080916.

SDS/PAGE, Western Blot – Test of HSP70 Ab on heat stressed shellfish for FISH441

Pacific oysters, a mussel, barnacles and a clam (sp. ?) were transferred from the holding tank to a large beaker with sea water which was placed into a 37C water bath. The shellfish were incubated in this water bath for ~3hrs. Tissues were collected from each, transferred to a 50mL conical tube and immediately placed in a dry ice/ethanol bath:

Oyster – gills, muscle and mantle

Clam – whole clam

Mussel – whole mussel

Barnacles – whole barnacles

0.02 – 0.07g of each tissue were weighed, added to a 1.5mL tube containing 0.5mL CelLytic MT + protease inhibitors and homogenized. For the barnacles, ~8 barnacles were transferred to a weigh boat and smashed with a hammer. This was then transferred to a 1.5mL tube with CelLytic MT + protease inhibitors. Tubes were spun @ max speed @ 4C for 10mins. Supe was transferred to a fresh 1.5mL snap cap tube. 15uL of each sample was transferred to a screw cap tube containing 15uL of 2X reducing sample buffer. Tubes were boiled for 5mins and then spun for 1min @ max speed @ 4C. Samples were loaded on Pierce 4-20% Tris-HEPES SDS PAGE gels and run 150V for 45mins. 10uL of SeeBlue Plus ladder was also loaded on the gel.

Gel, membrane, and blotting paper were soaked for 15mins in transfer buffer (Pierce recipe w/methanol). Proteins were transferred to nitrocellulose membranse for 30mins @ 20V. Gel was stained for ~40mins in Coomassie stain and then destained w/10% acetic acid solution until bands were clearly visible.

Western blotting was done according to Invitrogen WesternBreeze Chromogenic (anti-mouse) protocol. Primary HSP70 Ab was used at a 1:3000 dilution (per the Meistertzheim et al. paper).

SDS/PAGE, Western Blot – Test of new Western Breeze Kit & HSP70 Ab for FISH441

Ran varying amounts of Gigas Gill 24hr Control protein extract from 20080917. See table below:

Protein (ug) Volume of Sample (uL) Water up to 15uL (uL)
5 1.56 13.44
10 3.125 11.88
15 4.69 10.31
20 6.25 8.75
30 9.38 5.63
40 12.5 2.5
50 15.65

Volumes were adjusted to 15uL with H2O and combined with 15uL of 2X Reducing Sample Buffer in screw cap tubes. Also, 15uL of MSTN was mixed with 15uL of 2X Reducing Sample Buffer in a screw cap tube. This will serve as a positive control for the kit. Samples were boiled for 5mins. and then microfuged for 1min @ max speed @ 4C. Samples were loaded on Pierce 4-20% Tris-HEPES SDS PAGE gels and run 150V for 45mins. 10uL of SeeBlue Plus ladder and 10uL of the 12-tag positive control lysate were also loaded on the gel.

external image 20081231.JPG

Gel, membrane, and blotting paper were soaked for 15mins in transfer buffer (Pierce recipe w/methanol). Proteins were transferred to nitrocellulose membrane for 30mins @ 20V. Gel was stained for ~40mins in Coomassie stain and then destained w/10% acetic acid solution until bands were clearly visible.

After transfer, membrane was cut into two (one with the gigas samples and the other with the MSTN and the 12-tag lysate samples) and developed separately. Western blotting was done according to Invitrogen WesternBreeze Chromogenic (anti-mouse) protocol. Primary anti-HSP70 Ab was used at a 1:5000 dilution (2uL) and the primary anti-myc tag Ab (for MSTN) was used at a 1:1000 dilution (10uL). The anti-myc blot was allowed to develop for ~1min before stopping. The anti-HSP70 blot was allowed to develop for ~1.5hrs before stopping.

external image 20081231-01.JPGexternal image 20081231-02.JPG

Results:

 

The anti-HSP70 blot showed no bands. The anti-myc blot showed MSTN1b and positive control lysate bands. This confirms that the kit works properly.

RNA Gel – V. tubiashii mRNA samples (from 20081224)

external image 20081229.png

Lane 1 – Empty
Lane 2 – Total RNA, Control
Lane 3 – mRNA, Control
Lane 4 – Total RNA, Vibrio+gigas
Lane 5 – mRNA, Vibrio+gigas

Results:

rRNA removal seems to have worked relatively well. Still some residual rRNA present in the mRNA samples. Will submit 200ng of the Vibrio+gigas mRNA to HTGU for Illumina sequencing.