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.
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. 
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?
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.
Ran qPCR on DNased Abalone Dg RNA (07:12 Set), gDNA (06:50-10) and clean cDNA (from 20090422) using primers (H.crach_h-1fg_intron_Fw/Rv) designed to bind only to a region in an intron of the H.cracherodii hemocyanin gene. PCR setup/plate layout is here. Anneal temp of 50C was used.
Results: No PCR products in any samples, not even the positive control. It seems that the primers don’t work. Will design new primers, probably from a different species of abalone since there was essentially only one gene in H.cracherodii that had any intron sequence available..
Added 1/10 volume of 3M NaOAc (pH = 5.2) to sample (10uL) and then 2 volumes of ice cold 100% EtOH to samples (220uL). Mixed thoroughly and incubated O/N @ -20c.
Cells stored in EtOH were pelleted 5000g, 10mins, 25C. A brownish smear was present along the inside of the tube after spinning; not really a pellet per se. Supe was removed and cells were washed twice with 1X PBS. The smear was reduced to a pellet after the first wash in PBS. The second wash resulted in a slightly smaller pellet, but a pellet was present nonetheless before proceeding. Cells were subjected to an enzymatic lysis in 180uL of a TE/Triton X-100/lysozyme mixture as described in the Qiagen DNeasy Kit for Gram-Positive Bacteria (and for cells having substantial cell walls). Cells were incubated in this mixture for 30mins @ 37C. 25uL of proteinase K and 200uL of Buffer AL were then added and the mixture was incubated @ 70C for 30mins. Protocol then followed the “normal” steps for isolation of gDNA. Sample was eluted in 100uL of Buffer AE and then spec’d.
Results: Well, it doesn’t look like there is any DNA at all… Will try precipitating the sample and bringing up the DNA (if there’s any) in a small volume. It should be noted that I spec’d these samples using the “RNA” setting, so the constant for concentration calcs is wrong (40), but doing quick math using the DNA constant (50) shows that there’s still almost nothing there…
Set up qPCR with Cv_18s_F/R primers on the following samples that had previously come up negative in both reps using the diluted (1:20) cDNA that Mac had made specifically for the 18s runs:
3326A13
2100B07
3219A06
2100B15
3326A11
2100B12
2100A03
Plate layout/PCR set up is here. This is a second rep of these samples.
Results: Waters are clean. However, the following samples still are negative for an 18s amplicon:
A recent email from JGI indicates that they are satisfied with the quality of DNA (as seen on 20090601), however their estimate of the gDNA concentration (42ng/uL) means that we have ~16ug of DNA. They requested 50ug. Based on the gel, their calculations are reasonable. However, the NanoDrop suggests that are sample is ~1350ng/uL! So, I’ve respec’d the sample and did a few dilutions to see how it looked.
Results: The undiluted sample is approximately the same concentration as initially reported. A 1:1 dilution produces the expected concentration of half the undiluted. The 1:10 and 1:100 dilutions deviate a bit from the expected concentrations, but are reasonably close. I still don’t know how to explain the discrepancy between what the gel analysis suggests vs. the NanoDrop spectrophotometric data.
Set up qPCR with Cv_18s_F/R primers on the following samples that had previously come up negative in both reps using the diluted (1:20) cDNA that Mac had made specifically for the 18s runs:
3326A13
2100B07
3219A06
2100B15
3326A11
2100B12
2100A03
Plate layout/PCR set up is here.
Results: Waters are clean. However, the following samples still are negative for an 18s amplicon:
