Tag Archives: mantle

gDNA Isolation – Various gigas samples (continued from yesterday)

Pelleted residual tissue 10mins @ 10,000g @ RT. Transferred supe to new tubes. Precipitated DNA with 0.25mL 100% EtOH. Incubated 3mins @ RT. DNA was pelleted 5mins @ 5000g @ RT. Supe was removed, pellets were washed with 1mL 75% EtOH (x2). Supe was fully removed and the DNAs were resuspended in 300uL 8mM NaOH (made 7/9/10 SJW).

1M HEPES (provided with DNAzol) was added at a 1:100 dilution to achieve a pH = 8.0. This was based on the DNAzol protocol calculations (For 1mL of 8mM NaOH, use 101uL of 0.1M HEPES = pH 8.0).

Samples were spec’d on NanoDrop 1000. Used a sample with 8mM NaOH and 1M HEPES as a blank to match the pH = 8.0 of the samples.

Results:

260/280 ratios look good for all samples. Most of the samples have mediocre 260/230 ratios. Yields are excellent for all samples.

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gDNA Isolation – Various gigas samples

Placed ~20mg fragments of tissue in 250uL DNAzol. Added 1.35uL of Proteinase K (Fermentas; 18.5mg/mL) to reach a final concentration of 100ug/mL. Incubated RT, O/N, end-over-end rotation. Will complete DNA isolation tomorrow.

Sample List:

Vt Gigas Live #3 Gill 24E (from 20080828; Tatyana’s notebook)

Gigas Control #2 Gill 24E (from 20080828; Tatyana’s notebook)

NB-1209-10 (RNA Later)

SB-1209-14 (RNA Later)

WB-1209-09 (RNA Later)

0629 gill 5aza

0629 gonad 5aza

0629 mantle 5aza

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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.

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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!

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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…

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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.

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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.

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RNA Isolation – Sepia samples

Isolated RNA from 7 sepia samples received 20091125. Samples were removed from RNA Later, blotted and homogenized in 500uL of TriReagent. 500uL of additional TriReagent was added to the tubes after homogenization. Procedure was followed normally. The sepia retina RNA was isolated separately from the other samples and was resuspended in 100uL of 0.1% DEPC-H2O. The remaining samples were isolated and resuspended in 20uL of 0.1% DEPC-H2O. Nearly all samples had some sort of purple tint to them, ranging from almost black to extremely faint purple hue. The samples were spec’d and then stored @ -80C in Sam’s RNA Box #1.

Results:

Nearly all of the samples exhibited very strange curves and mediocre 260/280 ratios (for RNA). Could be due to the “purple stuff” carryover or possibly an effect of the RNA Later from which the samples were stored.

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Hard Clam Challenge – QPX Strain S-1 (continued from yesterday)

All clams appeared to be alive and well. Most had their siphons out when I arrived to start collecting tissues. Clams were shucked after 24hr challenge. Gill and mantle samples were collected in separate 1.5mL snap cap tubes, stored briefly on ice and transferred to -80C in “Hard Clam QPX Challenge 12/2/2009.” box.

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