Tag Archives: 16s

PCR – C.pugetti gDNA from 20090513 & 20090526

Previous PCRs from 20090601 & 20090602 both showed contamination in the negative control. Suspect that the primer stocks were contaminated due to the usage of older, non-sterile TE for reconstitution. New stocks were received and reconstituted with filter-sterilized TE. Working stocks were made with filter-sterilized Nanopure H2O. All pipettes, tips, tubes, racks were UV-sterilized in the biological hood. The PCR reaction was set up in the biological hood. PCR set up is here. Used universal 16s bacteria primers (27F, 1492R). Sequences from Sara Kelly. Anneal 60C.

Lane 1 – 100bp ladder

Lane 2 – gDNA (5/13/2009)

Lane 3 – gDNA (5/26/2009)

Lane 4 – H2O

Lane 5 – H2O

Lane 6 – H2O

Lane 7 – H2O

Results: Still contamination in the water-only samples!!!

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qPCR – Re-DNased abalone Dg RNA from earlier today

Done to verify removal of gDNA from RNA. Used H.crach_16s_syb_f/r primers. PCR workup/plate layout is here.

Results: Still f’ing gDNA! I’m pretty convinced that this is indeed due to the Ambion kit I’m using being old. Got mixed up with a newer kit, but neither had dates. Mac is going to be running a qPCR later today on DNased RNA that used the other Ambion kit. I will wait until the results of her qPCR to proceed.

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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.

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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.

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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.

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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.

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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.

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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.

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