Author Archives: kubu4

mRNA Isolation – hard clam gill #1 DNased RNA from today

DNased RNA from earlier today was split into four equal parts (175uL = 39.8ug). Three will be used for mRNA isolation and the fourth will remain as total RNA. Three of these were precipitated according to Ambion PolyAPurist Protocol: 1/10 volume 5M ammonium acetate, 1uL glycogen and 2.5 volumes of 100% EtOH. Incubated @ -80C for 30 mins. One sample was processed with the Promega PolyA Tract kit. The remaining two samples were processed according to PolyAPurist Protocol. Of those two, one of the samples was processed a second time to evaluate the effectiveness of running a sample through the PolyAPurist Protocol twice.

mRNA samples were precipitated O/N @ -20C according to the PolyAPurist Protocol.

RNA Precipitation – Hard clam gill #1 RNA from 20080819

DNAsed RNA using Ambion Turbo DNA-free kit, following the rigorous procedure. Diluted total RNA to 0.2ug/uL (Vf = 720uL). Added 1uL DNase and incubated the tube @ 37C for 30mins. Added an additional 1uL DNase and continued incubated for 30mins. Added 0.2 volumes of DNase Inactivation Reagent (158.4uL) and incubated at RT for 10mins with periodic mixing. Pelleted inactivation reagent according to protocol and transferred supe (DNA-free RNA) to clean tube.

Results: RNA looks really nice. Have a large quantity of RNA (700uL x 0.2275ug/uL = 159.25ug). Will split into four equal parts and isolate mRNA from 3 of the 4. Those three will be:

  1. Ambion kit x 1

  2. Ambion kit x 2

  3. Promega PolyA Tract kit.

qPCR – Repeat of qPCR from earlier today with fresh primer working stocks

This is an exact repeat of the qPCR from earlier today, but using a fresh working stock of the Vtub_16s_V3 primers. The plate layout/qPCR workup is here.

Results: Same as earlier today. Must be a bacterial contaminant somehwere that these 16s primers are picking up. Will order IGS primers that are species specific found in Lee et al. 2002.

qPCR – Repeat of 20090227 qPCR with clean water

This is an exact repeat of the qPCR from Friday, but using a fresh aliquot of water for preparation. The plate layout/qPCR workup is here.

Results: Same as Friday. Fluorescence comes up way too fast and there is contamination present in in the water. Will repeat with a fresh preparation of the primer working stocks.

qPCR – New 16s primers for V.tubiashii Control vs. Autoclaved gigas samples (see 20090224)

qPCR was performed using SensiMix/SYBR “kit” with DNAsed RNA samples from 20090224. This qPCR used the new V.tub_16s_V3 primers in hopes of getting better amplification; both in signal intensity and elimination of the double peak seen in the melting curves from 20090224. The plate layou/qPCR workup is here.

Results: Fluorescence comes up WAY too early; at like the 5th cycle! Also, there are two peaks in the melting curves. Additionally, there is a signal in the two water samples and the melting curve for this contamination matches up with one of the melting cure peaks seen in the actual sample melting curves. So, there is some sort of contamination somehwere. Will repeat this using a clean water for the master mix and hope the problem goes away.

qPCR – Replicate of V.tubiashii Control vs. Autoclaved gigas samples (see yesterday)

This is a repeat of the qPCR from yesterday, but without the 16s and OmpW primer sets due to double peaks in melting curves yesterday. Plate layout/qPCR workup is here.

Results: Similar to yesterday’s results, the amplification looks a bit odd when viewing on a log scale. However, the linear scale curves look to be normal. Melting curves look good for all genes examined and there is not any detectable gDNA in the RNA samples. Excellent…

qPCR – V.tubiashii Control vs. Autoclaved gigas samples (see below)

qPCR was performed using SensiMix/SYBR “kit” with DNAsed RNA samples and cDNA made earlier today. The plate layout/qPCR workup is here.

Results: Generally, the amplification looks a bit odd when viewing on a log scale. However, the linear scale curves look to be normal. There doesn’t appear to be any gDNA contamination in the RNA samples, BUT the Vtub_16sV2 primers used on the RNA samples also do not produce much of a signal in the cDNA either. The melting curves for the 16s and the Vtub_OmpW primer sets have multiple peaks. Will likely order new 16s primes, due to weak signal.

Will redo qPCR on the DNAsed RNA to make sure that the lack of detectable signal is due to lack of gDNA and NOT because the 16s primers don’t work. Also will repeat in order to have a replicate of the other samples.