Tag Archives: DNase

DNase – Hard Clam Gill RNA (from earlier today)

DNased 5ug of RNA from each sample with Ambion’s Turbo DNA-free Kit, according to the manufacturer’s rigorous protocol. Samples were spec’d and stored @ -80C in the “Hard Clam V.t. Experiment RNA” box.

Results:

Due to lack of a positive control (and lack of primers known to amplify gDNA), these DNased RNA samples will NOT be checked to verify elimination of gDNA carryover at this time. Will proceed with making cDNA for qPCR anyway.

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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.

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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.

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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.

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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.

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DNase – DNase C.gigas RNA from 20110120, 20110121 and 20110124

5ug of RNA was DNased using Ambion’s Turbo DNA-free kit, following the rigorous protocol (0.5uL of DNase for 30 mins then additional 0.5uL of DNase for 30mins). Calcs for DNase reactions are here. RNA was stored @ -80C in Shellfish RNA Boxes 4 and 5. Samples will be spec’d on Monday.

Results:

Overall, the RNA looks really good (based on OD 260/280 numbers). Not surprisingly, the OD 260/230 values for all samples dropped, likely due to the addition of the buffer (salts) used in the DNase reaction. Emma says she will check these samples for residual DNA.

–UPDATE (20110131)– Emma checked all DNased RNA samples on 20110131 using C.gigas 18s primers (SR ID: ?). She has not listed the results of the whether or not all samples are clean or if some still have residual gDNA carryover.

–UDPATE (20110201– Samples that appear to have residual gDNA carryover based on Emma’s qPCR on 20110131: Muscle C6, Gill 1hr C2 & E2.

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DNase – DNasing Hard Clam RNA from yesterday

Pooled 2ug of each sample in each group (MAX, CA, MA) for a total of 6ug of RNA (3 total samples), brought volume up to 50uL and DNased using Ambion’s Turbo DNA-free following the rigorous protocol. Calcs can be seen here. Spec’d:

Results:

All samples look pretty good. Oddly, the 260/280 ratios are absolutely perfect, despite the 260/280 ratios from each individual sample being less than stellar (see yesterday’s EtOH precipiation). Also of note is that the concentrations for all three samples are extremely close, reflecting the accuracy of the NanoDrop readings of each individual sample used for the pool as well as my pipetting. :)

RNA was stored @ -80C in “Sam’s RNA Box #1.”

Recovered ~50uL from each sample which means each pool yielded ~3.85ug of RNA after DNase treatment. Will proceed with making cDNA from these three pools. In the interest of time (and the failure of our Opticon), I will not verify that these do NOT still contain gDNA (and, it’s pretty unlikely that they do).

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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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DNase Treatment – Tim’s adult gigas challenge RNA (from 20090930)

Used 5uL of RNA from each sample, brought samples up to 50uL with H2O and treated according to Ambion’s Turbo DNA-free kit. Transferred treated samples to a PCR plate to facilitate further manipulation of the samples. Will perform qPCR on these samples to make sure treatment worked.

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