DNased RNA was used for cDNA rxns. Workup of the amount of RNA used in each rxn (472ng/uL) is here (Google Spreadsheet), along with previous cDNA batch numbers. Actual RT master mix set up is here. Samples were given to Lisa for qPCR analysis.
Tag Archives: black abalone
NanoDrop – H.crach DNased RNA (from 20090623)
Samples were first spec’d , since they had not been since their DNase treatement.
Sample 08:4-15 was spec’d later (accidentally skipped). [RNA] = 79.19ng/uL.
Calculations were made for cDNA rxns (see tomorrow’s RT rxns for workup).
qPCR – Abalone cDNA (07:12 set from 3/3/2009 by Lisa) and DNased RNA (from 20090623)
Now that we have a solid positive control, I’ll use the H.crach_h-1fg_intron primers to check the existing cDNA and DNased RNA. qPCR plate layout/set up is here. Anneal temp 50C.
Results: Looks like all the cDNA and DNased RNA are negative ! Finally! Will make cDNA from the DNased RNA.
qPCR – Abalone gDNA (H.crach 06:7-1)
This is to test (again!) the H.crach_h-1fg_intron primers and obtain a working positive control. This gDNA is from Lisa/Nate. Acquired from them 20090717. Don’t know date/method of isolation, but this should be good, recent gDNA. qPCR plate layout/set up is here. Anneal temp 50C. Ran serial dilutions of the gDNA: undiluted, 1:10 and 1:100.
Results: Signals look good. The undiluted comes up around 30 cycles. Should be able to use it as a positive control for gDNA detection PCRs.
qPCR – Abalone cDNA (07:12 set from 3/3/2009 by Lisa) and DNased RNA (from 20090623)
This is nearly a repeat of the qPCR earlier today due to the fact that the positive control never amplified. This is being done to check whether or not there is gDNA contamination in these cDNA and DNased RNA. Will use H.crach_h-1fg_intron primers. In hopes of remedying the positive control issue, I have used three sets of gDNA and used 5uL instead of the usual 1uL for their respective reactions. qPCR plate layout/set up is here. Anneal temp 50C.
Results: No detectable amplification in any gDNA sample. However, one sample did produce a melting curve peak, while no other samples did. Still doesn’t provide me with anything useable. Will get good gDNA from Freidman Lab ASAP.
qPCR – Abalone cDNA (07:12 set from 3/3/2009 by Lisa) and DNased RNA (from 20090623)
This is being done to check whether or not there is gDNA contamination in these cDNA and DNased RNA. Will use H.crach_h-1fg_intron primers. gDNA 07:12-15 was used as a positive control, based on results from yesterday’s qPCR. qPCR plate layout/set up is here. Anneal temp 50C.
Results: Everything came up negative, including the positive control! Also, the machine experienced an error at ~cycle 39, so no melting curve info. See below.
qPCR – Abalone gDNA
Used up the remainder of the one positive control gDNA that worked with all the primers in yesterday’s reaction (H.crach_h-1fg_intron, H.iris_actin_intron, H.crach_16s), so need to find a new set of gDNA to use for future positive controls. qPCR plate layout/set up is here. Anneal temp 50C. Used the following gDNA with :
06:50-9 – This was the good gDNA used as previous controls. Added 10uL of H2O to the tube in hopes of getting more useable DNA.
06:4-7 – No date/info available on tube.
07:12-15 – No date/info available on tube.
Results: Got decent signals with the H.crach_h-1fg primers for two of the three gDNAs. Will use the 07:12-15 gDNA as a positive control for tomorrow’s qPCR.
qPCR – Abalone RNA/DNased RNA & “dirty” and “clean” cDNA
This was done to really test the detection methods we’re using for gDNA contamination in our qPCRs. qPCR plate layout/set up is here. Anneal temp 50C.
It should be noted that the 07:12-08 RNA was diluted 1:10 before adding to the rxns to make it similar concentration to the DNased RNA. Also, ran out of the gDNA when adding to the 16s primer rxn. Used 1uL of H2O to “wash” the stock tube and added that in hopes of still detecting something. Unfortunately this was the only stock of H.crach gDNA that came up positive in yesterday’s rxns. “Dirty” cDNA is cDNA made with DNased RNA determined to still have gDNA using 16s primers AFTER the cDNA was already made. “Clean” cDNA used DNased RNA determined to be free of gDNA BEFORE the cDNA was made.
Results:
Signals were present with 16s primers in the following samples:
RNA 08:3-7, clean and dirty cDNAs and gDNA.
The other two primer sets only showed signals in the RNA 08:3-7.
Nothing came up in any of the DNased RNA samples.
qPCR – Abalone gDNA/cDNA
Due to lack of amplification in gDNA samples from 20090710 and 20090708 with either set of intron primers, will repeat with additional gDNA samples to make sure the primers are the problem and not the gDNA. Used the H.iris_actin_intron_Fw/Rv and the H.crach_h-1fg_intron_Fw/Rv primers. PCR setup/plate layout is here. Anneal temp 50C.
Results: Got a weak signal (C(t) ~ 37) in only the 06:50-9 rxns, but it did work with both primer sets.
qPCR – DNased Abalone Dg RNA from 20090625
Ran qPCR on gDNA (06:50-10) to test new primers (H.iris_actin_intron_Fw/Rv) designed to bind only to a region in an intron of the H.iris actin gene. Hopefully there’s enough homology between H.iris (primer source) and H.cracherodii (template source) for this to work. PCR setup/plate layout is here. Anneal temp 50C.
Results: No signal. 