Samples were submitted for sequencing. Mac prepped all Roberts Lab samples excluding the Opsin VMC gel slice 2 from 20091217-02. The gel slice was purified with Millipore spin columns. The sample was diluted 1:1 with H2O and submitted for sequencing, one time from each direction using the Sep_Op_Fw2/Rv2 primers. Plate layout can be found here on sheet labeled “20091223”.
qPCR – BB & DH cDNA (from earlier today)
qPCR was set up on these cDNAs using HMGP_5’/3′ and SPI_5’/3′ primers. qPCR set up and plate layout can be found here.
Results:
qPCR Data File (Opticon): 20091223_144042.tad
Data workup: PROPS BB_DH Gene Expression Miner Workup
Reverse Transcription – BB & DH DNased RNA (from 20090514)
Made a fresh, double batch (50uL rxn instead of 25uL) of cDNA according to Promega MMLV protocol using oligo dT primers. cDNA was put into a plate for faster qPCR loading. cDNA calcs and plate layout are here. Briefly, RNA and oligo dTs were combined, brought up to 37uL, heated @ 70C for 5mins and immediately placed on ice. RT master mix was made (RT master mix calcs are here), 13uL was distributed to each well. Samples were incubated @ 42C for 1hr and then 95C for 5mins.
UPDATE: cDNA plate was discarded 20120320 by SJW.
PCR – Sepia cDNA
This is an exact repeat of the PCR from yesterday, due to inconsistencies between repeated PCRs from yesterday and earlier today. Here’s yesterday’s workup
Results:
Gel Loading (from left to right):
Opsin Primers (lanes 2-10), Rhodopsin Primers (same loading order, lanes 12-20)
1 – 100bp ladder
2 – retina
3 – fin
4 – 4th arm
5 – dorsal mantle center
6 – dorsal mantle side
7 – ventral mantle center
8 – ventral mantle side
9 – H2O
10 – H2O
Opsin Primers
Single bands in retina, fin and ventral mantle center samples. Negative controls are clean. Bands will be excised and stored @-20C in Sam’s “Purified Inserts” box for eventual sequencing. Tubes are labelled with a “2” to differentiate them from the gel run earlier today.
Rhodopsin Primers
Single bands in retina and fin. Double bands in ventral mantle center sample. Second band is very faint is ~700bp. Negative controls are clean. Bands, including the faint 700bp VMC, will be excised and stored @-20C in Sam’s “Purified Inserts” box for eventual sequencing. Tubes are labelled with a “2” to differentiate them from the gel run earlier today.
PCR – Sepia cDNA
This is an exact repeat of the PCR from yesterday, due to inconsistencies between repeated PCRs from yesterday and earlier today. Here’s yesterday’s workup. Samples were stored @ 4C O/N after completion of PCR. Gell was run on 20091217.
Results: This is getting embarrassing. Opsin results are same as yesterday (good!), but Rhodopsin results are slightly different than yesterday’s AND the day before yesterday’s results!
Gel Loading (from left to right):
Opsin Primers (lanes 2-10), Rhodopsin Primers (same loading order, lanes 12-20)
1 – 100bp ladder
2 – retina
3 – fin
4 – 4th arm
5 – dorsal mantle center
6 – dorsal mantle side
7 – ventral mantle center
8 – ventral mantle side
9 – H2O
10 – H2O
Opsin Primers
We see a band in the retina, fin, 4th arm, & ventral mantle center samples as we did yesterday. This replicates yesterday’s PCR results, which is good. Negative controls are clean. Bands will be excised and stored @-20C in Sam’s “Purified Inserts” box for eventual sequencing. NOTE: This gel was run on 20091217 and the tubes are dated as such!
Rhodopsin Primers
A single band is present in the retina, dorsal mantle center and ventral mantle center samples. We see TWO bands in the fin sample. These results differ from yesterday’s in that yesterday, a single band was present ONLY in the retina and fin samples. Two days ago there was a single band in each of the retina and fin samples and two bands in the ventral mantle center sample. Bands will be excised and stored @-20C in Sam’s “Purified Inserts” box for eventual sequencing. NOTE: This gel was run on 20091217 and the tubes are dated as such!
I am going to repeat these PCRs again. I can’t stand the fact that I am getting such freaking inconsistent results in an extremely simple PCR. Aaargh!
PCR – Sepia cDNA and DNased RNA
Set up PCR on recent Sepia cDNA and DNased RNA samples using both S. officianalis_rhodopsin_F, R primers & Sep_op_F2, R2 primers. After yesterday’s intirguing PCR results, we need to confirm that the DNased RNA is free of contaminating gDNA. Additionally, we want to excise bands from the cDNA samples for sequencing. PCR set up is here. RNA was prepped as though making cDNA; diluted 0.2ug of DNased RNA in a final volume of 25uL. Used 1uL of this for PCRs.
Rhodopsin primers require 3mM Mg2+ in the PCR rxn and the opsin primers require 2mM Mg2+ in the PCR rxn. 25mM Mg2+ was added as required and is shown on the PCR set up link above.
Results: n00b alert! The gel below is a repeat of yesterday’s PCR, except with DNased RNA samples added to verify no gDNA carryover. However, you’ll notice that the PCR results on the cDNA don’t look identical to yesterday’s PCR. Ugh.
Gel Loading (from left to right):
Opsin Primers (top half of gel), Rhodopsin Primers (same loading order, bottom half of gel)
RNA samples (left sides of gel), cDNA samples (right sides of gel)
1 – 100bp ladder
2 – retina
3 – fin
4 – 4th arm
5 – dorsal mantle center
6 – dorsal mantle side
7 – ventral mantle center
8 – ventral mantle side
9 – H2O
Loading is repeated in the above order for the cDNA, which is the right halves of the gel.
Well, the good news is that no signal is seen in the DNased RNA samples, thus confirming that there is no detectable gDNA carryover.
Opsin Primers
We see a band in the retina, fin & ventral mantle center samples as we did yesterday. However, we see a band in the 4th arm sample, which was not present yesterday. We also do NOT see a band in the ventral mantle side sample like we did yesterday. Negative controls are negative.
Rhodopsin Primers
We see a band in the retina and fin samples as we did yesterday. However, we no longer see the dual bands in the ventral mantle center as were seen yesterday. Negative controls are negative.
Bands were excised from the gel, placed in 1.5mL snap cap tubes and stored in the -20C in Sam’s “Purified Inserts” box.
Because of the lack of repeated results, I will repeat this PCR for a third time, with just the cDNA again, to see if I can duplicate one set of results…
PCR – Sepia cDNA (from yesterday)
Set up PCR on recent Sepia cDNA samples using both S. officianalis_rhodopsin_F, R primers & Sep_op_F2, R2 primers. PCR set up is here. Looking back at my old paper (gasp!) notebook from March 2007, I noticed that each primer set required differing amounts of Mg2+. Rhodopsin primers require 3mM Mg2+ in the PCR rxn and the opsin primers require 2mM Mg2+ in the PCR rxn. Mg2+ was added as required and is shown on the PCR set up link above.
Results:
Gel Loading (from left to right):
Opsin Primers (lanes 2-10), Rhodopsin Primers (same loading order, lanes 12-20)
1 – 100bp ladder
2 – retina
3 – fin
4 – 4th arm
5 – dorsal mantle center
6 – dorsal mantle side
7 – ventral mantle center
8 – ventral mantle side
9 – H2O
10 – H2O
Opsin Primers
We see an intense band in the retina sample, as expected, since opsin is constitutively expressed in this tissue. We also see a band in the fin and both dorsal mantle samples.
Negative controls are blank.
Rhodopsin Primers
We see an intense band in the retina sample. We also see a band in the fin sample. We see two bands in the ventral mantle center tissue, possibly suggesting a rhodopsin isoform is also being expressed in this tissue.
Negative controls are blank.
Overall, these results are rather intriguing because they clearly demonstrate that both genes are being differentially expressed in non-eye tissue of Sepia officianalis.
Reverse Transcription – Abalone 07:12 DNased RNA (from 20090623)
Spec – DNased Abalone 07:12 RNA
Apparently, these samples had not been spec’d after DNase treatment.
Results:
Samples range in quality (260/280) from not great to perfect. Will perform calcs to make cDNA.
Set up reverse transcription rxns using 174ng of each DNased RNA (sample 07:12-04 was limiting; only 6.1uL available), using Promega oligo dT primers and M-MLV Reverse Transcriptase according to Promega protocol. RNA/oligo dT primer workup is here. Primer and DNAsed RNA were mixed and brought up to 18.25uL with H2O. Samples were heated @ 70C for 5mins and then placed immediately on ice. The RT Master Mix set up can be found here. 6.75uL of the RT master mix was added to each tube, mixed, spot spun, incubated 42C for 1hr., 95C for 3mins and then the samples were given to Lisa in the Friedman Lab.
Reverse Transcription – Sepia DNased RNA
DNase Treatment – Sepia RNA (from 20091204)
Samples were DNase treated with Ambion’s Turbo DNA-free kit, following the rigorous protocol. Used 6uL of each sample, brought up to 50uL with H2O, added 5uL of 10x buffer and 1.5uL of DNase. Incubated 37C for 30mins, added an additional 1uL of DNase and incubated @ 37C for 30mins. Added 0.2 volumes of DNase Inactivation reagent and incubated at RT for 2mins with regular mixing. Spec’d RNA.
Results:
Set up reverse transcription rxns using 200ng of each DNased RNA, using Promega oligo dT primers and M-MLV Reverse Transcriptase according to Promega protocol. RNA/Oligo dT primer workup here. Primer and DNAsed RNA were mixed and brought up to 18.25uL with H2O. Samples were heated @ 70C for 5mins and then placed immediately on ice. The RT master mix set up can be found here. 6.75uL of the RT master mix was added to each tube, mixed, spot spun and then incubated @ 42C for 1hr, heat inactivated @ 95C for 3mins and stored @ 4C.
Bioanalyzer – Herring Liver cDNA for SOLiD Libraries
Samples were run on the DNA 1000 chip for cDNA smear analysis.
Results: 2 of the 4 samples (2L & 3L) look perfect (<20% of cDNA in the 25-150bp range). 6L has <20% of the cDNA in the 25-150bp range (which is perfect), but exhibits a “smear” from ~250-500bp. cDNA in this range suggests overamplification, which will skew gene expression profile. Can repeat PCR for 6L using outer gel slices and reduce the number of cycles to prevent overamplification, if desired. Spoke to Steven and since these samples won’t be used to evaluate gene expression (they’re for SNP discovery), we won’t worry about it for the time being.
Sample 4L has some very strange signals being generated in the ~500-800bp range. Additionally, the virtual gel image (not shown) shows a great deal of smearing, unlike the other samples.
