Isolated RNA in 1mL of Tri-Reagent according to the manufacturer’s protocol. Also, finished RNA isolation of samples that were started 20110506. Samples were resuspended in 50uL 0.1%DEPC-H2O and spec’d.
Results:
Tag Archives: Vibrio tubiashii
RNA Isolation – Hard Clam Gill Tissue from Vibrio Experiment (see Dave’s Notebook 5/2/2011)
Isolated RNA in 1mL of Tri-Reagent according to manufacturer’s protocol. Samples were precipitated with isopropanol and stored over the weekend @ -20C. Will conclude isolation on Monday. The samples isolated were:
MA 1-11
MA Vt 1-11
SOLiD Sequencing Submission
Submitted the following 8 samples for SOLiD sequencing at HTGU:
| SB unmeth C.gigas | C.gigas | gill pool | gDNA |
| SB meth C.gigas | C.gigas | gill pool | gDNA |
| MA | Mercenaria mercenaria | gill pool | polyA(x2) |
| BX | Mercenaria mercenaria | gill pool | polyA(x2) |
| Vt RE22 | Vibrio tubiashii (RE22) | gDNA | |
| Vt STRAIN | Vibrio tubiashii (ATCC 19106) | gDNA |
qPCR – HpaII/MspI Digests from earlier today
Restriction Digests – Various gigas gDNA from earlier today
Digest master mixes are here. Digests were incubated @ 37C for 2hrs. and then heat inactivated @ 80C for 20mins.
gDNA Isolation – Various gigas samples (continued from yesterday)
Pelleted residual tissue 10mins @ 10,000g @ RT. Transferred supe to new tubes. Precipitated DNA with 0.25mL 100% EtOH. Incubated 3mins @ RT. DNA was pelleted 5mins @ 5000g @ RT. Supe was removed, pellets were washed with 1mL 75% EtOH (x2). Supe was fully removed and the DNAs were resuspended in 300uL 8mM NaOH (made 7/9/10 SJW).
1M HEPES (provided with DNAzol) was added at a 1:100 dilution to achieve a pH = 8.0. This was based on the DNAzol protocol calculations (For 1mL of 8mM NaOH, use 101uL of 0.1M HEPES = pH 8.0).
Samples were spec’d on NanoDrop 1000. Used a sample with 8mM NaOH and 1M HEPES as a blank to match the pH = 8.0 of the samples.
Results:
260/280 ratios look good for all samples. Most of the samples have mediocre 260/230 ratios. Yields are excellent for all samples.
gDNA Isolation – Various gigas samples
Placed ~20mg fragments of tissue in 250uL DNAzol. Added 1.35uL of Proteinase K (Fermentas; 18.5mg/mL) to reach a final concentration of 100ug/mL. Incubated RT, O/N, end-over-end rotation. Will complete DNA isolation tomorrow.
Sample List:
Vt Gigas Live #3 Gill 24E (from 20080828; Tatyana’s notebook)
Gigas Control #2 Gill 24E (from 20080828; Tatyana’s notebook)
NB-1209-10 (RNA Later)
SB-1209-14 (RNA Later)
WB-1209-09 (RNA Later)
0629 gill 5aza
0629 gonad 5aza
0629 mantle 5aza
qPCR – V.tubiashii primers test (Vpt A and Vt IGS)
Utilized to sets of primers obtained from the Friedman Lab: VptA (referred to as “Hasegawa”, even though the reference article calls the primers Vtp A) and Vt IGS (referred to as “Lee” primers, presumably from a published article). For template, used “RE22 DNA” that was given to me by Elene. Tube is dated 9/10/09 and has no indication of concentration. Performed qPCR on a set of 10-fold dilutions. Plate layout/qPCR set up is here, along with dilution series used.
Results:
The VptA primer set generated a nice looking set of dilutions with appropriate spacing (~3.2 Ct/10-fold dilution). HOWEVER, the raw fluorescence signal is very low (only 0.4 units; good signal is usually 3-5-fold higher) AND the melting curve doesn’t look that great. The melting curve could look poor due to the low signal, since it doesn’t come up much higher than background levels.
It should be noted that the low fluorescence levels generated could simply be due to the amplicon size generated by these primers. The amplicon size is only 63bp. An amplicon of this size might not be able to incorporate significant amounts of dye to generate a “normal” level of fluorescence (1.25 – 2 units).
The Vt IGS primers failed to generate any product.
qPCRs – Tim’s adult gigas challenge cDNA (from 20091009)
Set up qPCR with Cg_P450 primers and TNFRAF3’/5′ primers. Plate layout/setup is here.
Results: Processed with PCR Miner. Normalized to EF1. Standard Error bars. Here is spreadsheet with workup.
P450: The two control samples (AC & CC) show significant difference in expression between the Air and CO2 treated samples. The Vibrio treated samples (AV & CV) show no difference in expression between Air and CO2 treatments.
The Air samples (AC & AV) show significant difference in expression between the Control (AC) and Vibrio treated (AV) samples.
TNFRAF3: Expression levels of the Vibrio treated samples (AV &CV) were too low for Miner processing.
There are significant differences in expression between the Air (AC) and CO2 treated (CC) samples.
Set up qPCR with Cg_IkB primers and Cg_Prx6 primers. Plate layout/setup is here.
Results: Processed with PCR Miner. Normalized to EF1. Standard Error bars. Here is spreadsheet with workup.
IkB:
No difference in expression between Air (AC & AV) and CO2 (CC & CV) treatments.
Appears to be a significant difference between the Air Control (AC) and the Air Vibrio treated (AV) samples.
Prx6:
Significant difference in expression between Air Control (AC) and CO2 Control (CC) samples, but no difference in the Vibrio treated samples.
Significant difference in expression between the Air Control (AC) and the Air Vibrio treated (AV) samples, but no difference in the CO2 treated Vibrio samples (CC & CV).
qPCR – Tim’s adult gigas challenge cDNA (from 20091009)
Set up qPCR with Cg_HIF1 (hypoxia induced factor 1) primers and prostaglandin E2 primers. Plate layout/setup is here.
Results: Processed with PCR Miner. Normalized to EF1. Standard Error bars. Here is spreadsheet with workup.
HIF1:
No significant differences between any treatments.
Prostaglandin E2:
No significant differences between any treatments.