Streaked C. pugetti onto marine broth plate + a few biphenyl crystals and incubated at RT over the weekend.
Results: No growth as of Monday, 20090427.
Author Archives: kubu4
Reverse Transcription – cDNA from DNased Abalone RNA from 20090420
Prepared cDNA using an equal amount of RNA from all samples (442.6ng). This amount was based on using the maximum allowable volume of RNA for the RT rxn AND the sample with the lowest [RNA] (08:3-20; 24.59ng/uL). cDNA was prepared according to the Promega MMLV RT recommendations. Here is the work up for the cDNA rxns. cDNA was stored @ -20C.
qPCR – Abalone DNased RNA from yesterday
Performed qPCR to evaluate gDNA removal w/ 2x Immomx and SYTO 13. qPCR/plate set up is here.
Results: The two cDNA samples come up as positive. No flourescence detected in any other gamples. However, melting curves look suspicous despite the fact that the “Quantitation” view indicates now amplification.
Bacteria – C. pugettii culture CONTINUED (from 20090419)
Transferred 1mL of the culture to a 50mL conical containing 4mL of 1x Marine Broth and a couple crystals of biphenyl. Kept the cap loosened and incubated at 20C with shaking at 200RPM. Kept the existing culture in the incubator as well. No apparent growth.
gDNA Removal – Abalone RNA from 20090402 and 20090331
Transferred 50uL of RNA to fresh tubes and processed them using the Ambion Turbo DNA-free kit according to the manufacturer’s protocol.
Bacteria – C. pugettii culture
Rehydrated ATCC isolate according to directions. Added 5mL of 1x Marine Broth to a glass culture tube. Used 1mL of this to rehydrate ATCC sample and then added back into the culture tube. Added a few crystals of biphenyl, which had been exposed to UV for ~5mins prior. Incubated at 20C, no shaking. This was done at ~11:30AM.
Sequencing – Dungan isolates
Submitted two plates for sequencing. Each sample two times from each direction.
PCR – Two new Dungan isolates
Repeat of yesterday’s PCR, but with AmpliTaq, less gDNA and 50uL rxn volume. PCR set up is here.
Gel loaded and run by Steven. Not entirely sure of the loading order. However, it doesn’t really matter…
Results: Still absolutely nothing.
UPDATE: Noticed on 20090713 that the reactions didn’t have dNTPs. Probably explains why it didn’t work!
qPCR – Rab7_SYBR primers on abalone RNA and DNased RNA
Attempt to find out if gDNA contamination exists iafter Ambion treatment. Previous test (on 20090414) suggests the QT Kit did not eliminate gDNA. PCR set up and plate layout here. Used Immomix and SYTO 13.
PCR – Two new Dungan isolates from earlier today
Set up PCRs on:
MIE-14y
VNTc-1.2-C1/G10
Used Euk A/B and LABY A/Y primers. Anneal temp 50C. PCR set up is here.
NOTE: Due to the extremely low concentrations of gDNA from these two samples, I used a large amount of gDNA in the rxns. Check the PCR set up link for actual numbers.
Lane 1 – Hyperladder (5uL)
Lane 2 – VNTc-1.2-C1/G10 (Euk primers)
Lane 3 – MIE-14y (Euk primers)
Lane 4 – H2O (Euk primers)
Lane 5 – H2O (Euk primers)
Lane 6 – VNTc-1.2-C1/G10 (Laby primers)
Lane 7 – MIE-14y (Laby primers)
Lane 8 – H2O (Laby primers)
Lane 9 – H2O (Laby primers)
lane 10 – 100bp Ladder
Results: Nada. Probably because of low [gDNA], but could also be due to PCR inhibitors in the gDNA. Will retry using Amplitaq and less gDNA.