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.
Tag Archives: SYTO13
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 – Abalone RNA, check for gDNA
Abalone RNA isolated from this week was qPCR’s with 16s primers to determine if gDNA contamination was present. PCR/plate layout is here.
Results: All samples appear to have gDNA contamination. Will use Qiagen QT Kit for cDNA.
qPCR – Virginica cDNA (see workup sheet below for more info regarding cDNA)
Set up qPCR using the following Virginica primers provided by Mac:
Cv_BgBL
Cv_BGBP
Cv_CysB
Cv_HMG
Cv_HSP70
Here is the qPCR workup sheet .
