Isolated gDNA from the following two samples:
MIE-14y
VNTc-1.2-C1/G10
Samples were spun @ 16,000g @ RT for 2mins. No visible pellets in either sample. EtOH was removed. “Pellets” were washed in 1x PBS (pH=7.6) two times and then the Qiagen DNEasy Kit protocol was followed. Samples were incubated @ 55C with Proteinase K for ~2hrs.
Results: Both samples show really, really low quantities of gDNA.
Anneal 55C. PCR set up is here (bottom half of sheet).
Lane 1 – Hyperladder
Lane 2 – gDNA
Lane 3 – cDNA pool
Lane 4 – No RT 06:5-31
Lane 5 – No RT 06:6-43
Lane 6 – No RT 08:3-5
Lane 7 – No RT 08:3-6
Lane 8 – No RT 08:3-7
Lane 9 – H2O
Lane 10 – H2O
Lane 11 – 100bp ladder
Results: No signal in the gDNA. Appropriate sized band in cDNA pool. Nothing in the water samples. HOWEVER, got bands in tow of the “No RT” rxns (08:3-6/7)!! It’s odd that the gDNA didn’t produce any signal, but there shouldn’t be any signal in the No RT rxns. This indicates gDNA contamination. Should have also tested RNA and DNased RNA. Will test these in order to determine if that system is better at eliminating gDNA carryover.
Used higher annealing temps to improve primer specificity, compared to yesterday’s results. PCR set up and plate layout is here.
See the PCR/plate set up link for samples. Hyperladder is placed between every 12 samples.
Results: See this Google Spreadsheet for a summary of the 4 gels from the last two days.
Used 3 sets of reverse primers:
Bay_Actin_Rv0
Bay_Actin_Rv2
Sea_Actin_Rv2
Primers were slected based on information from Steven’s notebook (#8, 12/30/2007-1/3/2008). Anneal temp 53C.
PCR set up here . Plate layout here .
Samples were run out by Steven the following day.
Gel 1 of 3
Gel 2 of 3
Gel 3 of 3
Results:
gDNA was isolated using 500uL of 10% Chelex. Samples were heated @ 95C for 1hr w/periodic vortexing. Samples were then spun 16,000g for 5mins @ 4C. Stored @ -20C in Bay x Sea Scallop Project Box.
Performed a new PCR on the three types of samples listed above with new TOLLIP primers. The new TOLLIP primers (H.discus_806_F/Ab_866_Rv) surround a putative intron. Thus, they will be useful for determining the presence of gDNA contamination in RNA/cDNA. Anneal temp 55C. PCR set up is here .
Lane 1 – Hyperladder
Lane 2 – gDNA
Lane 3 – cDNA (QT)
Lane 4 – RNA (untreated)
Lane 5 – DNased RNA
Lane 6 – QT Kit, No RT
Lane 7 – H2O
Lane 8 – H2O
Results: This could possibly be the most confusing gel I’ve ever had the “pleasure” of running/analyzing, despite that it only has 7 samples.
No band in the gDNA sample, which could be explained by the intron size being too large for amplification with a basic polymerase. The cDNA worked as expected and contains a band of ~150bp. The RNA sample has a faint band of ~750bp. The DNased RNA sample has a band of ~400bp. The differences seen betweeen the gDNA (Lane 2), RNA (Lane 4) and DNased RNA (Lane 5) are truly bizarre. The No RT sample has no band. And, to top things off, one of the H2O samples is blank , but the other one has a band of ~1500bp! Ugh. How is all of this even possible?
Steven had me re-PCR the old Dungan isolates with the new EukA/B primers. Anneal temp 50C. PCR set up here (bottom half of sheet) .
NOTE: Sample #30 was not in the rack of tubes that Steven gave to me.
Results:
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.
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 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.
