Tag Archives: reverse transcriptase

Reverse Transcription – Ronit’s C.gigas DNased ctenidia RNA

Proceeded with reverse transcription of Ronit’s DNased ctenidia RNA (from 20181016).

Reverse transcription was performed using 100ng of each sample with M-MMLV Reverse Transcriptase from Promega.

Briefly, 100ng of DNased RNA was combined with oligo dT primers and brought up to a final volume of 15uL. Tubes were incubated for 5mins at 70oC in a PTC-200 thermal cycler (MJ Research), using a heated lid. Samples were immediately placed on ice.

A master mix of buffer, dNTPs, water, and M-MMLV reverse transcriptase was made, 10uL of the master mix was added to each sample, and mixed via finger flicking. Samples were incubated for 1hr at 42oC in a PTC-200 thermal cycler (MJ Research), using a heated lid, followed by a 5min incubation at 65oC.

Samples were stored on ice for use later this afternoon by Ronit.

Samples will be stored in Ronit’s -20oC box.

Reverse transcription calcs (Google Sheet):

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Reverse Transcription – O.lurida DNased RNA 1hr post-mechanical stress

Performed reverse transcription on the Olympia oyster DNased RNA from the 1hr post-mechanical stress samples from Jake’s project. To accommodate the large numbers of anticipated genes to be targeted in subsequent qPCRs, I prepared 100μL reactions (normally, 25μL reactions are prepared) using 250ng of each DNased RNA. A 1:10 dilution of the oligo dT primers (Promega) was prepared to improve pipetting accuracy. All incubations were performed in a thermal cycler without using a heated lid.

DNased RNA was combined with NanoPure H2O and oligo dT primers in 24 wells of a PCR plate, heated @ 70C for 10mins and immediately placed on ice. After 5mins, the plate was spun 2000g @ RT for 2mins and returned to ice.

25.25μL of a master mix containing 5x M-MLV Buffer (Promega), dNTPs (10mM each; Promega), and M-MLV Reverse Transcriptase (50U/rxn; Promega) was distributed to each well and mixed via pipetting. The plate was heated @ 42C for 1hr, 95C for 3mins. The plate was spun 2000g @ RT for 2mins and then stored @ -20C.

Plate layout and all calculations can be found here (Google Sheet): 20150806_Jake_oly_mech_stress_cDNA_calcs

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Reverse Transcription – Subset of Jake’s O.lurida DNased RNA

Currently don’t have sufficient reagents to perform reverse transcription on the entire set of DNased RNA (control and 1hr.heat-shocked O.lurida ctenidia samples). To enable Jake to start testing out some of his primers while we wait for reagents to come in, Steven suggested I generate some cDNA for him to use.

Used the following DNased RNA:

  • HC1
  • NC1
  • SC1
  • HT1 1
  • NT1 1
  • ST1 1

Reverse Transcription Calcs: 20150522_Jake_Oly_cDNA_Calcs

Briefly:

  • Reactions run in 0.5mL snap cap tubes
  • 250ng of DNased RNA used in each reaction
  • Combined DNased RNA with oligo dT primers and water; incubated 70C 5mins; immediately placed on ice
  • Added 6.75μL of buffer/dNTP/enzyme master mix to each sample; incubated 42C for 1hr; 95C for 3mins

Samples will be given to Jake and stored @ -20C.

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