Vibrio samples (2-1 and 2-2) from 20090212 were submitted to the mass spec facility.
Tag Archives: Vibrio tubiashii
Trypsin digestion – Vibrio 2D spots CONTINUED (from yesterday)
Sample prep was continued from yesterday. SpeedVac’d final, pooled elutions for 1.5hrs. Stored tubes @ -80C. Will submit samples 2-1 and 2-2 for mass spec analysis.
Trypsin digestion – Vibrio 2D spots from 20081217
Samples were prepared according to Goodlett Lab protocol and incubated O/N on LabQuake.
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
Vibrio challenge CONTINUED (from yesterday)
One of the two starter cultures from yesterday were used to inoculate 500mL 1x LB+1% NaCl and incubated O/N @ 30C 200RPM.
Vibrio challenge
2X 10mL starter cultures of in 1x LB+1% NaCl were inoculated with two separate colonies of V. tubiashii and incubated O/N @ 30C 200RPM.