Tag Archives: 16s

PCR – C.pugetti DNA from 20090513 & 20090526

Performed PCR as set up here using universal bacterial 16s primers (sequences provided by Sara Kelly).

Lane 1 – 100bp ladder

Lane 2- 5/13 DNA

Lane 3 – 5/26 DNA

Lane 4 – H2O

Results: Contamination in the water sample. Hopefully this is just bad technique (never thought I’d say that about myself) and not general, bacterial contamination in the primer stocks, since the stocks (nor the PCR rxns) were prepared steriley. Will repeat.

qPCR – Check DNased abalone RNA (by Lisa) for gDNA

qPCR was performed with 16s_sybr primers on the DNased RNA that Lisa did. Annel temp 55C. Sample set up and plate layout is here.

Results: Still got signals in all of the samples, including the waters. Personally, I think the primers are contaminated or are forming crazy dimers. Lisa came by and picked up cDNA to run other genes on.

qPCR – Repeat (modified) of yesterday’s abalone cDNA check

qPCR was performed with 16s_sybr primers on a subset of the “No RT” cDNA rxns from yesterday at both 55C and 60C. Sample set up and plate layout is here.

Results: Still getting signals in the “No RT” rxns and possibly in the waters. Cut run short to start another. Will test Lisa’s previously DNased RNA.

qPCR – Abalone cDNA (QT) from earlier today

qPCR was performed with 16s_sybr primers on a subset of the cDNA rxns and all of the “No RT” rxns from earlier to detect the presence of contaminating gDNA. qPCR was also performed with the Rab7_sybr primers on a subset of the cDNA rxns to check that the assay would work. The qPCR set up sheet/plate layout is here. Annealing temp = 55C.

Results: Apparently the gDNA wipeout step did NOT work! Also, some signal in the water samples.

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…