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.
Author Archives: kubu4
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.
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.
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)
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.
rRNA Removal – V. tubiashii total RNA from yesterday
rRNA removal was continued from O/N precipitation. Processed the samples according to the Ambion MICROBExpress Kit protocol and resuspended final pellets in 25uL of The RNA Storage Solution. Samples were spec’d on the NanoDrop.
Samples were stored @ -80C in Sam’s RNA Box #1.
RNA Isolation – V. tubiashii from challenge (see 20081216)
Added 10mL TriReagent to 2 x 50mL pellets (5.63 x 10^11 total bacteria; see calcs on 20081219) from the control samples collected on 20081219. Added 10mL TriReagent to 1 x 50mL pellet (1.835 x 10^12 total bacteria; see calcs on 20081219) from the V.tubi + oyster samples collected on 20081219. Scaled rest of RNA protocol to match. Resuspended pellets in 100uL 0.1%DEPC-H2O. Samples were NanoDropped.
The V. tubi + gigas sample was eventually diluted to contain 400uL (see final reading for that sample above).
RNA samples were precipitated O/N @ -20C according to Ambion protocol.
Vibrio challenge CONTINUED (from yesterday)
Significantly more bacteria in the container containing autoclaved oysters. Collected 2 x 50mL from each treatment. Collected ~750mL from each treatment. Cells were pelleted 4000RPM, 15mins, 4C. Supe was removed and pellets frozen @ -80C.
100uL of a 1:1,000,000 dilution of the control bacteria were plated on 1x LB + 1% NaCl plates and incubated O/N @ RT. 100uL of a 1:10,000,000 dilution of the exposed bacteria were plated on 1x LB + 1% NaCl plates and incubated O/N @ RT. Control colony count the next day = 563 colony forming units (CFU). Exposed colony count the next day = 367 CFU.
Control Calculations
563 CFU/100uL = 5.63 CFU/uL
5.63 CFU/uL x 1:1,000,000 dilution = 5.63 x 10^6 CFU/uL
5.63 x 10^6 CFU/uL x 1000uL/mL = 5.63 x 10^9 CFU/mL
Exposed Calculations
367 CFU/100uL = 3.67 CFU/uL
3.67 CFU/uL x 1:10,000,000 dilution = 3.67 x 10^7 CFU/uL
3.67 x 10^7 CFU/uL x 1000uL/mL = 3.67 x 10^10 CFU/mL
Vibrio challenge CONTINUED (from yesterday)
500mL culture was split evenly between two containers containing 3L sterile sea water each. One container also contained 3 large, autoclaved C. gigas. Containers had an air stone to promote circulation. 4 x 1mL samples were collected from each container, pelleted @ 10,00RPM 1min. Supe removed and samples stored @ -80C. Samples will be collected @ t = 0, 0.5, 1.0 and 24 hrs. Containers were covered with aluminum foil to minimize splashing caused the by the air stone.
100uL of a 1:1,000,000 were plated on 1x LB + 1% NaCl plates and incubated O/N @ 37C. Colony count the next day = 399 colony forming units (CFU).
399 CFU/100uL = 3.99 CFU/uL
3.99 CFU/uL X 1:1,000,000 dilution = 3.99 x 10^6 CFU/uL
3.99 x 10^6 CFU/uL x 1000uL/mL = 3.99 x 10^9 CFU/mL
3.99 x 10^9 CFU/mL x 250 mL/container = 9.975 x 10^11 CFU/container