PCR – Bay/Sea scallop hybrids

Because Rony’s results from her PCR did not match her previous results for the positive controls, I ran the PCRs myself on the controls and the hybrids. Anneal temp 50C. PCR set up, samples, etc. are here.

Results: The positive controls still do not match the results Rony got in two consecutive PCR attempts. However, the Bay_Actin_Rv2 and Sea_Actin_Rv3 primers do result in clearly distinguishable differences between bay and sea scallop gDNA. Lane 2 (bay gDNA/bay actin primer) has a large, ~1000bp band and lane 7 (sea gDNA/sea actin primer) produces a ~300bp band. Unfortunately, this does provide us with any useful info. Something needs to be reworked (i.e. possibly new target gene) in order to start getting some useful results.

However, the results we did get definitely confirm that the hybrids are NEITHER hybrids nor bay scallop, due to the fact that no bands are present in any other sample than the bay scallop gDNA samples.

PCR – Dungan isolates from 20090402 with Euk primers

Did PCR with new Euk primers designed by Steven. Should be one step higher taxonomically. PCR set up is here. Aneal temp 50C.

Lane 1 – Hyperladder

Lane 2 – 19t

Lane 4 – 13t

Lane 6 – 17t

Lane 7 – 100bp ladder

Lane 8 – 1.5t

Lane 10 – 1.2t

Lane 11 – Hyperladder

Lane 12 – 11t

Lane 14 – H5

Lane 15 – 100bp ladder

Lane 16 – 12t

Lane 18 – H2O

Lane 19 – H2O

Laen 20 – Hyperladder

Results: The new EukA/B primers worked wonderfully. The brightest band in each lane was excised and purified using Millipore DA spin columns. These will be stored and sequenced at a later date.

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.

cDNA – Abalone RNA from 20090331 & 20090402

cDNA was made from the above RNA samples using the Qiagen Quantitect RT Kit. The samples were laid out in a PCR plate. 274.2ng of RNA was used in the rxn for each sample, based on the lowest concentration RNA sample (08:3-20) to equalize all the samples. The Genomic Wipeout step of the kit requires 2uL of Genomic Wipeout enzyme/buffer to be added to 12uL of an RNA sample, so the calculations were done and can be found here. A check mark on the calculation sheet indicates that the water and then the RNA was added to the appropriate wells. Those with two check marks were used for a “No RT” rxn and thus, have duplicate wells (see plate layout).

The plate was mixed, spot spun, uncubated at 42C for 2mins and immediately placed on ice.

The RT and No RT master mixes were set up on ice and then added to the respective wells (see sheet here).

UPDATE: cDNA plate was discarded 20120320 by SJW.

PCR – New Dungan isolates

Repeat of PCR from 20090403, but using AmpliTaq and 50C annealing temp. PCR set up is here.

Lane 1 – Hyperladder

Lane 2 – 19t

Lane 3 – 100bp ladder

Lane 4 – 13t

Lane 6 – 17t

Lane 8 – 1.5t

Lane 9 – Hyperladder

Lane 10 – 1.2t

Lane 12 – 11t

Lane 13 – 100bp ladder

Lane 14 – H5

Lane 16 – 12t

Lane 17 – 100bp Ladder

Lane 18 – H2O

Lane 19 – H2O

Lane 20 – Hyperladder

Results: Nothing amplified! Possibly due to age of polymerase (?); over a year old. Will wait to repeat for new primers to arrive (EukA/B).

PCR – New Dungan isolates

Ran PCR with GoTaq on the new Dungan isolate gDNA from yesterday. PCR set up is here. 53C annealing temp.

Lane 1 – Hyperladder

Lane 2 – 17t

Lane 4 – H5

Lane 6 – 12t

Lane 7 – Hyperladder

Lane 8 – 1.5t

Lane 10 – 1.2t

Lane 12 – 11t

lane 13 – Hyperladder

Lane 14 – 13t

Lane 16 – 19t

Lane 18 – H2O

Lane 19 – Hyperladder

Lane 20 – H2O

Results: All the bands present are a bit larger than 400bp. However, the bottom band in the H5 sample is larger than any band present in any other samples. Additionally, the largest band in the H5 sample is between 800-1000bp. Bands were cut out from H5 (two bands: Top, Bottom), 1.5t, 1.2t, 13t. These will be purified and sequenced.

It’s also interesting to note that the bands present in this gel are found in the same samples as the last time this analysis was done. See Kevin’s Notebook, 20090212.