Category Archives: PROPS

PROPS

qPCR – C.gigas BB/DH cDNA for PROPS

Performed qPCR using cDNA from 20110311. This was performed for additional reps for TIMP3(BB) (SR IDs:1067 & 1106) and HMGP (SR IDs:359 & 360). Master mix calcs are here. Plate layout, cycling params, etc can be found in the qPCR Report (see Results).

Results:

qPCR Report (PDF)

qPCR Data File (CFX96)

Will analyze with PCR Miner and incorporate with previous PCR rep done for PROPS with these two genes. Oddly, samples in wells B09 and H09 have weird melt curves. As such, these samples will be excluded from analysis.

Share

qPCR – Check DNased RNA BB01 for Residual gDNA (from earlier today)

Ran qPCR on DNased RNA from earlier today to verify removal of contaminating gDNA. Used C.gigas 18s primers (SR IDs: 156, 157). 0.5uL (~40ng) of DNased RNA was used for testing. This corresponds, roughly, to the amount of sample that would be carried through to qPCR analysis of cDNA, assuming 1ug of RNA was used to make the cDNA (cDNA = 1000ng RNA/25uL = 40ng/uL, 1uL of cDNA in 25uL qPCR reaction). Positive control sample was ~25ng BB16 gDNA (from 20090519). Master mix calcs are here. Plate layout, cycling params, etc can be found in the qPCR Report (see Results). RNA was stored @ -80C in “Sam’s -80C Box”.

Results:

qPCR Report (PDF)

qPCR Data File (CFX96)

Residual gDNA is present in the sample. So, it’s become apparent that it’s virtually impossible to rid the BB01 RNA of contaminating gDNA. Will discuss with Steven and Mac if it’s feasible to exclude this from the additional PROPS analysis that needs to be done and how this could potentially affect our data. Talked to Steven and, duh, we can just remove the previous BB01 data from our analysis. Will make new batch of cDNA from existing DNased RNA samples.

Share

DNase – C.gigas BB01 (PROPS) RNA (from 20090507)

Since the previous DNase treatment failed for this sample, will repeat but will start with less RNA (5ug instead of 10ug). Need more DNased RNA to finish repeating of PROPS. Some samples had insufficient quantities of DNased RNA remaining in BB01. Used 5ug of RNA and followed Ambion’s “rigorous” protocol, utilizing a total of 2uL of DNAse for each sample. Briefly, samples were incubated @ 37C for 30mins, an additional 1uL of DNase was added to each sample, mixed and incubated for an additional 30mins @ 37C. After finishing protocol, samples were spec’d.

DNase Rxn Calcs:

BB01 (1.824ug/uL): 5ug/1.824ug/uL = 2.74uL RNA + 42.26uL H2O (to 45uL) + 5uL 10X DNase Buffer = 50uL

Results:

RNA looks OK, based on OD260/280. Would like that value to be higher, though. OD260/230 is low, which is typical post-DNased treatment. Will check for residual gDNA via qPCR.

Share

qPCR – Check DNased RNA BB01 for Residual gDNA (from earlier today)

Ran qPCR on DNased RNA from earlier today to verify removal of contaminating gDNA. Used C.gigas 18s primers (SR IDs: 156, 157). 0.75uL (~50ng) of DNased RNA was used for testing. This corresponds, roughly, to the amount of sample that would be carried through to qPCR analysis of cDNA, assuming 1ug of RNA was used to make the cDNA (cDNA = 1000ng RNA/25uL = 40ng/uL, 1uL of cDNA in 25uL qPCR reaction). Positive control sample was ~25ng BB16 gDNA (from 20090519). Master mix calcs are here. Plate layout, cycling params, etc can be found in the qPCR Report (see Results). RNA was stored @ -80C in “Sam’s -80C Box”.

Results:

qPCR Report (PDF)

qPCR Data File (CFX96)

Well, this sucks. Still gDNA contamination. Will just start with original RNA again and discard this “DNased” sample.

Share

DNase – C.gigas BB01 from 20110225

Used EtOH precipitated BB01 RNA from 20110225 and followed Ambion’s “rigorous” protocol, utilizing a total of 2uL of DNAse. Briefly, samples were incubated @ 37C for 30mins, an additional 1uL of DNase was added to each sample, mixed and incubated for an additional 30mins @ 37C. After finishing protocol, samples were spec’d.

Results:

RNA looks good, based on the OD260/280. As usual after DNasing, the OD260/230 is on the low side. Will check for residual gDNA via qPCR.

Share

Ethanol Precpitation – DNased RNA BB01 (from earlier today)

Due to residual gDNA contamination, will EtOH precipitate in order to treat with DNase again. Add 0.5 vols 3M NaAOc (pH=

5.2), 2.5 vols 100% EtOH, mixed and incubated @ -20C for 30mins. Pelleted RNA @ 16,000g, 4C 30mins. Washed RNA with 1mL 70% EtOH (2x due to fear of residual salts from DNase Buffer). Pelleted RNA @ 16,000g, 4C, 15mins. Resuspended RNA in 45uL nuclease-free H2O. Sample was stored @ -80C (in “Sam’s RNA Box #1) until it could be DNased again.

Share

qPCR – Check DNased RNA BB01 for Residual gDNA (from earlier today)

Ran qPCR on DNased RNA from earlier today to verify removal of contaminating gDNA. Used C.gigas 18s primers (SR IDs: 156, 157). 0.5uL (50ng) of DNased RNA was used for testing. This corresponds, roughly, to the amount of sample that would be carried through to qPCR analysis of cDNA, assuming 1ug of RNA was used to make the cDNA (cDNA = 1000ng RNA/25uL = 40ng/uL, 1uL of cDNA in 25uL qPCR reaction). Positive control sample was ~25ng BB16 gDNA (from 20090519). Master mix calcs are here. Plate layout, cycling params, etc can be found in the qPCR Report (see Results). RNA was stored @ -80C in “Sam’s -80C Box”.

Results:

qPCR Report (PDF)

qPCR Data File (CFX96)

Ugh. Still gDNA present in this sample. Hmmmm. Will consider starting from original RNA, but will precipitate this sample again and treat again to see if I can get rid of that cursed gDNA.

Share