Set up the MMLV RT rxns with random primers using ~833ng DNAsed RNA (prepared yesterday) according to the Promega MMLV Product Insert. This procedure is slightly different than what is in our lab protocol for RT rxns. Here is the workup for the rxns. cDNA was stored @ -20C in the “Vibrio” box.
Author Archives: kubu4
RNA Isolation – V.tubiashii samples from autoclaved gigas exposure (from 20081218)
RNA was isolated from the Control and V.tub+gigas samples from the 0, 1, & 24hr time points using 1mL TriReagent. No visible pellets. Used 20uL of 0.1%DEPC-H2O to resuspend RNA. Incubated @ 55C, 5mins. Spec’d.
Results: RNA looks OK, but not great. For the “V.tub + gigas t=1″ sample, the third spec reading is correct. The first two had the air bubble error.
DNAse Treatment – V.tubiashii total RNA (see above)
1ug of RNA in a volume of 12uL was DNAsed using the Ambion DNA-free Kit according to their protocol. RNA was transferred to a fresh tube and stored @ -80C in Sam’s RNA Box #1.
Epigenetics Experiment – Gigas treatment
Set up two containers with 3L seawater and 4 gigas in each container. The treated sample contained:
5mL of 5N NaOH
5mL of Bl
10mL of 1x LB
10mL of 95% EtOH
1mL of formamide
Both containers were covered, stored in the fume hood and incubated over the weekend. Experiment was started at 3:15PM.
Sample Submission – V.tubiashii Mass Spec
Vibrio samples (2-1 and 2-2) from 20090212 were submitted to the mass spec facility.
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.
mRNA – Submission for Agilent Bioanalyzer
Submitted 4uL (108ng/uL) of mRNA from gigas gill (from DATE!; sometime in late August) to the Center for Array Technologies (CART) at UW. Sample was labelled “SR01″.
Results:
Results were received on 20090210. Comment from CAT staff member was that it looked “pretty good.” Here are links to the gel (look for SR01), the electoropherogram of SR01, and the EGRAM of the ladder.
mRNA – Precipitation continued from yesterday
Samples were pelleted and washed with 70% EtOH according to Ambion PolyA Purist protocol. Pellets were resuspended in 10uL of The RNA Storage Solution (included in the Ambion PolyA Purist Kit).
Results: The gill mRNA looks great! Good yield and good ratios. Hemocyte mRNA looks kinda rough and a very low yield (which was to be expected).
mRNA Isolation – Hard Clam gill and hemo RNA
mRNA was isolated according to Ambion PolyA Purist protocol. After mixing samples with resin, samples were incubated @ RT for 1hr. Samples were washed per the protocol. However, the hemo sample was not clearing from the spin columns with the protocol-directed 3 min. spins. The column had to be spun up to 15 mins. in order for the column to clear. 
Results: The gill mRNA looks good (~1.8ug). The concentration of the hemo sample is extremely low and is below the error threshold for the NanoDrop, but it may not be that bad. The samples are in a relatively large volume (~200uL) and the yield is expected to be very small. So, I will precipitate these samples O/N @ -20C according to Ambion PolyA Purist protocol in order to concentrate them.
RNA – Hard clam hemo RNA (from 20090121)
The two RNA samples from yesterday were precipitated and washed according to the Ambion PolyA Purist protocol and resuspended in 50uL of 0.1% DEPC-H2O.
Results: RNA readings look better than they did prior to precipitation. The hemo RNA samples will be combined with previous hemo RNA samples and mRNA will be isolated using the Ambion PolyA Purist Kit.