Author Archives: kubu4

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.

PCR – Dungan isolate (MIE-14v) gDNA from 20090708

PCR of MIE-14v just to make sure that we can’t get a product from this sample, despite NanoDrop readings suggesting that there’s no DNA. Used both LABY and Euk primer sets. PCR set up is here. Anneal temp 50C.

Lane 1 – 100bp Ladder

Lane 2 – Euk

Lane 3 – Euk H2O

Lane 4 – Euk H2O

Lane 5 – Euk H2O

Lane 6 – LABY

Lane 7 – LABY H2O

Lane 8 – LABY H2O

Lane 9 – LABY H2O

Lane 10 – 100bp Ladder

Results: Nothing, as expected. Need to devise a new method of isolating gDNA from these “problem” isolates.

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. :(

DNA Precipitation CONTINUED – Dungan MIE-14v gDNA from yesterday

Sample was pelleted by spinning in a microcentrifuge @ max speed, 4C for 30mins. Supe was removed and sample CAREFULLY washed with 1mL 70% EtOH. Sample was spun in a microcentrifuge @ max speed, 4C for 10mins. Supe was removed, sample brought up in 10uL of TE and spec’d.

Results: Nothing. Absolutely no DNA in this sample at all. It’s odd that the Qiagen Kit procedure (even without the lysozyme treatment) has worked on all the other Dungan isolates, but not this one. I wonder if the EtOH storage is having an effect on the cells; lysing them for some reason? Maybe the cells should be sent to us in culture medium instead?

PCR – Bay/Sea Scallop DNA

An additional attempt to get the actin primers to work for use in screening samples for bay/sea scallop hybrids. The scallop_actin_fw primer was used in conjunction with the following:

bay_actin_Rv0 (Rxn 1)

bay_actin_Rv2 (Rxn 2)

sea_actin_Rv4 (Rxn 3)

sea_actin_Rv5 (Rxn 4)

PCR set up is here. Just used Bay or Sea scallop gDNA (chelexed). When/If get this working correctly, will start screening the hybrid samples. Anneal of 53C.

Lane 1 – 100bp Ladder

Lane 2 – Rxn 1: Bay

Lane 3 – Rxn 1: Sea

Lane 4 – Rxn 1: H2O

Lane 5 – Rxn 1: H2O

Lane 6 – Rxn 2: Bay

Lane 7 – Rxn 2: Sea

Lane 8 – Rxn 2: H2O

Lane 9 – Rxn 2: H2O

Lane 10 – Rxn 3: Bay

Lane 11 – Rxn 3: Sea

Lane 12 – Rxn 3: H2O

Lane 13 – Rxn 3: H2O

Lane 14 – Rxn 4: Bay

Lane 15 – Rxn 4: Sea

Lane 16 – Rxn 4: H2O

Lane 17 – Rxn 4: H2O

Lane 18 – 100bp Ladder

Results: Rxn 1 shows amplification with both Bay & Sea Scallop gDNA. The bands are close in size, but look like they would be more distinguishable if run on higher percentage gel and for a longer period of time to get better separation. However, there is contamination in one of the two water samples..

Rxn 2 shows amplification of only the Bay Scallop gDNA.

Rxn 3 shows amplification in both Bay & Sea Scallop gDNA and both bands are of the exact same size.

Rxn 4 shows no amplification in either set of gDNA.

Using the primers used in Rxn 1 will probably allows us to succesfully screen potential hybrids. Just need to remember to use high-percentage agarose gels and run samples for longer periods of time to get sufficient separation.