Tag Archives: BB

DNase – C.gigas BB01 from 20110216

Used EtOH precipitated BB01 RNA from 20110216 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:

First reading had an air bubble and should be ignored. DNased RNA looks good, based on 260/280 ratios. As is usually the case for DNased RNA, the 260/230 ratios are on the low side. Will check DNased RNA for residual gDNA.

Ethanol Precipitation – 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.

qPCR – Check DNased RNA BB01 & 09 (from earlier today)

Ran qPCR on DNased RNA from earlier today to verify that it was free 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).

There is residual gDNA in the BB01 sample. Will EtOH precipitate and treat again.

DNased BB09 was stored @ -80C in “Mac’s Gigas DNased RNA Box #1″ (on the top shelf) with the rest of the PROPS DNased RNA.

DNase – C.gigas BB/DH (PROPS) RNA (from 20090507)

Need more DNased RNA to finish repeating of PROPS. Some samples had insufficient quantities of DNased RNA remaining in BB01 and BB09. Used 10ug of each 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): 10ug/1.824ug/uL = 5.48uL RNA + 39.52uL H2O (to 45uL) + 5uL 10X DNase Buffer = 50uL

BB09 (0.506ug/uL): 10ug/0.506ug/uL = 19.77uL RNA + 25.23uL H2O (to 45uL) + 5uL 10X DNase Buffer = 50uL

Results:

260/280 values look great. 260/230 values look bad, but this is not unusual for samples post-DNase treatment.

qPCRs – Mac’s BB/DH cDNA from 20091223

GNRR2 and CALL primer sets used. These are duplicates based on initial differences seen between BB and DH expression. qPCR set up and plate layout is here.

Results:

qPCR Data File (Opticon): 20100111_180230.tad

Data workup: PROPS BB_DH Gene Expression Miner Workup

 

SPI and CP17A primer sets used. These are duplicates based on initial differences seen between BB and DH expression. qPCR set up and plate layout is here.

Results:

qPCR Data File (Opticon): 20100111_141711.tad

Data workup: PROPS BB_DH Gene Expression Miner Workup

 

EF1 primers in duplicate, since I had not done these primers yet. qPCR set up and plate layout is here.

Results:

qPCR Data File (Opticon): 20100111_102001.tad

Data workup: PROPS BB_DH Gene Expression Miner Workup

 

qPCR – BB & DH cDNA (from 20091223)

qPCR was set up on these cDNAs using the following primers:

FP008495.p.cg.6 (“GSTO1″, “Glutathione S-transferase omega-1″) – This was upregulated in BB SOLiD data.

These were run in duplicate to take up a full PCR plate. qPCR set up and plate layout can be found here.

Results:

qPCR Data File (Opticon): 20091231_152520.tad

Data workup: PROPS BB_DH Gene Expression Miner Workup

qPCRs – BB & DH cDNA (from 20091223)

qPCR was set up on these cDNAs using the following primers:

AJ582629.p.cg.6 (“DEF1″, “Defensin 1″) – This was upregulated in BB SOLiD data.

CB617519.p.cg.6 (“RETST”, “All-trans retinol”) – This was upregulated in BB SOLiD data.

qPCR set up and plate layout can be found here.

Results:

qPCR Data File (Opticon): 20091230_173747.tad

Data workup: PROPS BB_DH Gene Expression Miner Workup

 

 

qPCR was set up on these cDNAs using the following primers:

CU684779.p.cg.6 (“SEMSA”, “Semaphorin-SA”) – This was upregulated in BB SOLiD data.

FP004879.p.cg.6 (“TIMP3″, “Metalloproteinase inhibitor 3″) – This was upregulated in BB SOLiD data.

qPCR set up and plate layout can be found here.

Results:

qPCR Data File (Opticon): 20091230_143643.tad

Data workup: PROPS BB_DH Gene Expression Miner Workup

qPCR – BB & DH cDNA (from 20091223)

qPCR was set up on these cDNAs using the following primers:

FP010108.p.cg.6 (“DJB12″, “DnaJ homolog subfamily B member 12″) – This was upregulated in DH SOLiD data.

AJ565670.p.cg.6 (“TOP1″, “DNA topoisomerase 1″) – This was upregulated in BB SOLiD data.

qPCR set up and plate layout can be found here.

Results:

qPCR Data File (Opticon): 20091229_164912.tad

Data workup: PROPS BB_DH Gene Expression Miner Workup

qPCR – BB & DH cDNA (from 20091223) and Emma primer sets for testing

qPCR was set up on these cDNAs using the following primers:

EW778389.p.cg.6 (“DPGN”, “Serine protease inhibitor dipetalogastin”) – This was upregulated in DH SOLiD data.

FP001672.p.cg.6 (“PGSC1″, “Peptidoglycan-recognition protein SC1a/b”) – This was upregulated in DH SOLiD data.

Included three primer sets of Emma’s (matrillin, beta tub and chaperonin). These were set up with no template and done in duplicate.

qPCR set up and plate layout can be found here.

Results:

qPCR Data File (Opticon): 20091229_133148.tad

Data workup: PROPS BB_DH Gene Expression Miner Workup

Emma’s “beta tub” primers show some weird fluorescence, however none of the primer sets show any thing in the melting curve analysis.