Tag Archives: QPX

Chloroform Clean Up – Lexie’s QPX RNA from 20110504

After submission of QPX samples to HTGU for Illumina library prep yesterday, I was notified that there was insufficient RNA for the QPX RNA samples. I checked the source RNA on the Roberts Lab NanoDrop1000 and determined that they had high phenol contamination (large peak at 270nm), which results in a large exaggeration in the OD260 absorbance (NanoDrop1000 report[JPEG]; notice terrible OD260/280 ratios; did not save screen shot of absorbance peaks.). As such, the concentrations that Lexie had listed in her notebook for these samples are highly inaccurate and highly inflated. To remove the phenol, I brought all of her QPX RNA samples from 20110504 up to ~200uL with 0.1%DEPC-H2O, added 200uL of chloroform, vortexed for 30s, spun at 12,500g RT for 15mins, and transferred aqueous phase to new tube. Then performed an ethanol precipitation on the aqueous phase. Added 0.1 vols of 3.0M sodium acetate (pH = 5.2), 2.5 vols of 100% EtOH, mixed and incubated at -20C for 1hr. Pelleted RNA by spinning at 16,000g 4C for 15mins.

Results:

As suspected, most of these samples have absolutely no RNA in them. However, the samples that do (the “Control” samples), look great! Pooled 2ug each of the RT Control a & b samples and pooled 2ug each of the 10C Control a & b samples (which are ATCC). Calculations are here. Will take them down to HTGU tomorrow to replace the bad samples that were provided yesterday.

Share

qPCR – Lexie’s QPX Temp & Tissue Experiment (see Lexies Notebook 4/26/2011)

Ran qPCR with Lexie’s cDNA samples from this experiment with the following primer sets in order to better evaluate her biological reps:

QPX_SPB_F/R (SR ID: 387, 388)

LABY_A/Y (SR ID: 116, 121)

LABY was run as a potential normalizing gene. Master mix calcs are here. Plate layout, cycling params, etc. can be found in the qPCR Report (see Results). Samples were run in duplicate and were labeled according to what was written on the tops of Lexie’s cDNA tubes.

Results:

qPCR Data File (BioRad CFX96)

qPCR Report (PDF)

LABY primers worked, but the melt curves don’t look that good. I’ll let Lexie worry about the rest of the analysis.

Share

QPX Washes

Washed 4 day old QPX cultures. 3 isolates (S-1, TD-81, ATCC), two flasks (13mL) of each were washed in the following manner:

  1. Each flask’s contents were transferred to a 50mL conical tube.

  2. Each tube was topped with sterile sea water and mixed by inversion numerous times.

  3. Tubes were centrifuged 10mins, 3000g at RT.

  4. Transferred mucus and pellet to empty 50mL conical tube.

  5. Topped with sterile sea water and mixed by inversion numerous times.

  6. Centrifuged 10mins, 3000g at RT.

  7. Removed as much supe/mucus as possible without disturbing the pellet.

  8. Topped with sterile sea water and mixed by inversion numerous times.

  9. Repeat steps 7 through 8 until sample is mucous-free and the pellet can be resuspended.

Pellets were eventually resuspended in 1mL of sterile sea water, split between two 1.5mL snap cap tubes and then brought up to 1mL with sterile sea water.

Share

Hard Clam Challenge – QPX Strain S-1 (continued from yesterday)

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.

Share

Hard Clam Challenge – QPX Strain S-1

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.

Share