Performed reverse transcription on DNased RNA from the hard clam vibrio tubiashii challenge experiment (see Dave’s Notebook 5/2/2011), following the Promega M-MLV RT protocol with ~1ug of DNAsed RNA. Master mix calcs are here. Reactions were done in a plate. cDNA was diluted 1:4 with H2O.
Performed RT on DNased RNA using Promega MMLV RT and Oligo dT according to manufacturer’s protocol, using 1ug of DNased RNA. Due to large number of samples, cDNA was made in PCR plate. Plate layout and calcs are here. cDNA was diluted 4-fold (to 100uL total volume) based on qPCR done by Emma on 20110202.
Prepared cDNA using 1ug of RNA from each of the 3 pools (CA, MA, MAX) and processed according to Promega’s M-MLV protocol, using oligo dT primers. Calcs and master mix set up are here. Briefly, RNA was combined with oligo dT primers, denatured @ 70C for 5mins, immediately placed on ice for 2mins, mixed with RT master mix, incubated 1hr @ 42C, 3mins @ 95C, and then stored @ -20C.
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).
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.
Spec – DNased Abalone 07:12 RNA
Apparently, these samples had not been spec’d after DNase treatment.
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.
Ligation reactions from yesterday were subject to reverse transcription according to protocol. Master mix workup info is here. Samples were incubated @ 42C, 30mins and then cleaned up using the Qiagen MiniElute PCR Purification Kit, according to the Ambion WTK protocol.
After RT rxn, samples were run on a Novex 6% TBE-Urea gel according to Ambion WTK protocol. Samples were loaded, left to right: 2L, 3L, 4L, 6L. Ladders are to the left of each sample. The break in the smear in the 6L sample is a tear in the gel.
The recommended range of cDNA (100-200bp) were excised from the gel and were cut into four pieces, according to the Ambion WTK protocol. The two outer gel slices from each sample will be stored @-20C. Then proceeded to the emulsion PCR. Here is an image of the gel after the cDNA was cut out:
The four samples from yesterday were prepared according to the Agilent SOLiD WTK protocol. Briefly:
Results: All four samples appear to have cDNA. Interestingly, the “Amped cDNA trout RBC control ribo(-)” sample was the sample that had no detectable RNA after fragmentation, BUT this sample produced the highest yield of cDNA… See below.
1.5uL of each sample was transferred to a 0.5mL snap cap tube and stored @ -80C in the “Samples for Bioanalyzer” box for submission on the DNA 1000 Chip.
The Yellow/Brown plot above is the “Amped cDNA trout RBC poly I:C ribo(-) & polyA” sample and exhibits a strange profile at the 220-230nm range that differs than the three other samples.