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.
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…
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.
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.
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.