Tag Archives: lake trout

Gel Purification & PCR cDNA SOLiD Libraries – Abalone, Yellow Perch, Lake Trout, Herring

cDNA was gel purified according to Ambion’s Whole Transcriptome Analysis Kit. The appropriate regions (100 – 200bp) were excised and cut in to 4, 1x5mm pieces. The two “internal” pieces were transferred to individual PCR tubes. The “outer” pieces were transferred together to a 1.5mL snap cap tube and stored @ -20C.

Three images are below. The first two are the gels before excising the 100 – 200bp region of the gel. The third is the image of the SECOND gel after the specified region was excised. An image was not taken of Gel 1 after excision (whoops!).

Gel 1

 

Gel 2

NOTE: The WB sample in the gell above is actually a yellow perch sample, NOT an abalone sample!

 

Gel 2 AFTER EXCISION

NOTE: The WB sample in the gell above is actually a yellow perch sample, NOT an abalone sample!

 

In-gel PCR SOLiD Libraries – Abalone, Yellow Perch, Lake Trout, Herring

In-gel PCR was performed on the individual “internal” gel pieces that were excised, as described below from earlier today. PCR rxns/cycling were performed according to Ambion’s Whole Transcriptome Analysis Kit. PCR ran O/N. PCR master mix set up is here (bottom half of sheet).

Share

Reverse Transcription SOLiD Libraries – Abalone, Yellow Perch, Lake Trout, Herring

Samples were speedvac’d to dryness and resuspended in 3uL of nuclease-free H2O. Samples were then reversed transcribed according to Ambion’s Whole Transcriptome Analysis Kit. RT master mix set up is here (top portion of sheet).

Share

Hibridizaton/Ligation SOLiD Libraries – Abalone, Yellow Perch, Lake Trout, Herring

All 8 samples were hybridized/ligated according to Ambion’s Whole Transcriptome Analysis Kit using Adaptor A.

Share

Bioanalyzer for SOLiD Libraries – Fragmented mRNA from Perch, Lake Trout & Herring RNA samples

1uL of each sample from 20100325 was run on the Agilent 2100 Bioanalyzer on a RNA Pico 6000 chip to evaluate RNA quantity and fragmentation.

Results:

Share

SOLiD Library Prep – mRNA (perch, lake trout, herring from 20100318) Fragmentation

Fragmented mRNA according to Ambion’s Whole Transcriptome Sequencing Kit. Cleaned up sample using Ribominus Concentration Module (Invitrogen) according to Ambion’s WTS Analysis Kit. Samples were eluted w/20uL of H2O and stored @ -80C. Will Bioanalyze and speedvac at a later date.

Share

Bioanalyzer for SOLiD libraries – Total and mRNA from Perch, Lake Trout & Herring RNA samples (CONTINUED from yesterday)

Total and mRNA aliquots (~5ng/uL) were run on the Agilent Bioanalyzer Pico RNA chips.

Results:

The gel below shows the comparison/results of total RNA and subsequent mRNA isolations. The gel indicates the following:

  1. The HPWS09 total RNA (Herring) is totally degraded, but shows the expected profile in the mRNA prep. It would be extremely interesting to see if the degradation has any effect on sequencing, as the mRNA will get fragmented any way in the next step of library construction.

  2. mRNA isolations worked for all samples. Although one might be inclined to say that mRNA isolation did NOT work for the WB sample, one has to take in to consideration that the gel software adjusts the gel contrast to enhance low signals. That’s why all the mRNA samples exhibit a dark background. mRNA generates a broad, relatively weak signal when compared to a total RNA sample. So, the software attempts to boost the low signal for display purposes. Thus, if we were to decrease this signal boosting (or contrast) for the WB mRNA so that the background color matched the WB total RNA background color (white), the rRNA bands visible in the WB mRNA sample would fade to a point where they would not be visible. See the electropherogram overlay (below the gel) for a more visual comparison of this concept.

Electropherogram Overlays of WB total RNA and WB rRNA

The WB total RNA is the red graph which shows extremely high levels of rRNA (as expected). After subsequent mRNA isolation (the blue graph), the rRNA is virtually gone and no longer comprises a significant portion of the sample.

Share

mRNA Precipitation for SOLiD – Perch, Lake Trout, & Herring mRNA (CONTINUED from yesterday)

mRNA was pelleted and washed according to Ambion’s MicroPolyA Purist Kit. Pellets were resuspended in 8uL nuclease-free H2O and spec’d. 0.5uL was taken from each sample, transferred to a fresh tube, diluted to ~5ng/uL and stored @ -80C for eventual Bioanalyzer analysis. mRNA samples were stored @ -80C until we receive the Ribominus Concentration Module Kit from Invitrogen (turns out we didn’t have any!) for cleaning up the RNA after fragmentation.

Results:

Overall, this mRNA doesn’t look that great. However, I did notice that all samples had (to varying degrees) particulate matter that wouldn’t dissolve. Prior to spec’ing, the particulate matter was pelleted so as to not interfere. All samples will continue to be prepped for SOLiD analysis despite poor 260/280 ratios and low yields.

Share

mRNA Isolation for SOLiD – Perch, Lake Trout, and Herring total RNA

Received pooled lean and siscowet RNA from Rick. Samples will be processed immediately for SOLiD fragment libraries. Two 1.5mL snap cap tubes labelled:

L.T. 2ug muscle sisco pool

L.T. 2ug muscle lean pool

RNA was first precipitated according to the Ambion MicroPolyA Purist Kit protocol (0.1 vol 5M ammonium acetate, 1uL glycogen, 2.5 vols 100% EtOH). Samples were incubated @ -80C for 30mins. Samples were resusupended in 250uL nuclease-free H2O and spec’d.

 

Starting quantities PRIOR to total RNA precipitation:

Perch Samples:

WB tRNA (WB tRNA ~12ug 24.5uL)

PQ tRNA (PW tRNA ~12ug 21.88uL)

CT tRNA (CT tRNA ~12ug 25uL)

Herring:

1 G/O HPWS09 (20ug)

Lake Trout:

L.T. 20ug muscle sisco pool

L.T. 20ug muscle lean pool

Removed 1uL of each sample, diluted to ~5ng/uL and stored @ -80C to run on the Bioanalyzer.

Used Ambion MicroPolyA Purist Kit according to protocol. Samples were treated twice to ensure elimination of rRNA from the samples. After second run through MicroPolyA Purist, samples were EtOH precipitated O/N @ -20C according to Ambion’s MicroPolyA Purist protocol.

Share

Sequencing – Dungan Isolates, Lake Trout HRM and Emma DD cloning

Submitted 1.5 plates for Sanger sequencing. Dungan isolates prepared by me, Lake Trout HRM prepared by Rony and Emma’s differential display cloning samples prepared by her. All primers were prepped by me. See the sequencing log for samples and plate layout.

Share