Tag Archives: dessication

qPCRs – Ronit’s C.gigas ploidy/dessication/heat stress cDNA (1:5 dilution)

IMPORTANT: The cDNA used for the qPCRs described below was a 1:5 dilution of Ronit’s cDNA made 20181017 with the following primers! Diluted cDNA was stored in his -20oC box with his original cDNA.

The following primers were used:

18s

  • Cg_18s_F (SR ID: 1408)

  • Cg_18s_R (SR ID: 1409)

EF1 (elongation factor 1)

  • EF1_qPCR_5′ (SR ID: 309)

  • EF1_qPCR_3′ (SR ID: 308)

HSC70 (heat shock cognate 70)

  • Cg_hsc70_F (SR ID: 1396)

  • Cg_hsc70_R2 (SR ID: 1416)

HSP90 (heat shock protein 90)

  • Cg_Hsp90_F (SR ID: 1532)

  • Cg_Hsp90_R (SR ID: 1533)

DNMT1 (DNA methyltransferase 1)

  • Cg_DNMT1_F (SR ID: 1511)

  • Cg_DNMT1_R (SR ID: 1510)

Prx6 (peroxiredoxin 6)

  • Cg_Prx6_F (SR ID: 1381)

  • Cg_Prx6_R (SR ID: 1382)

Samples were run on Roberts Lab CFX Connect (BioRad). All samples were run in duplicate. See qPCR Report (Results section) for plate layout, cycling params, etc.

qPCR master mix calcs (Google Sheet):

RESULTS

No analysis here. Will analyze data and post in different notebook entry. This entry just contains the qPCR setup, resulting data, and a glimpse of how each primer performed.

Nothing is broken down based on sample ploidy or experimental conditions.

18s

qPCR Report (PDF):

qPCR File (PCRD):

qPCR Data (CSV):

Amplication Plots

Melt Curves

DNMT1

qPCR Report (PDF):

qPCR File (PCRD):

qPCR Data (CSV):

Amplication Plots

Melt Curves

EF1

qPCR Report (PDF):

qPCR File (PCRD):

qPCR Data (CSV):

Amplication Plots – Manual Threshold (Linear)

Amplication Plots – Manual Threshold (Log)

Amplication Plots – Automatic Threshold (Linear)

Amplication Plots – Automatic Threshold (Log)

Melt Curves

HSC70

qPCR Report (PDF):

qPCR File (PCRD):

qPCR Data (CSV):

Amplication Plots

Melt Curves

HSP90

qPCR Report (PDF):

qPCR File (PCRD):

qPCR Data (CSV):

Amplication Plots

Melt Curves

Prx6

qPCR Report (PDF):

qPCR File (PCRD):

qPCR Data (CSV):

Amplication Plots

Melt Curves

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):

qPCR – Ronit’s DNAsed C.gigas Ploidy/Dessication RNA with elongation factor primers

After I figured out the appropriate DNA and primers to use to detect gDNA in Crassostrea gigas samples, I checked Ronit’s DNased ctenidia RNA (from 20181016) for residual gDNA.

Elongation factor primers:

  • EF1_qPCR_5′ (SRID 309)
  • EF1_qPCR_3′ (SRID 310)

BB16 from 20090519 was used as a positive control.

Samples were run on Roberts Lab CFX Connect (BioRad). All samples were run in duplicate. See qPCR Report (Results section) for plate layout, cycling params, etc.

qPCR master mix calcs (Google Sheet):

Results

qPCR Report (PDF):

qPCR File (PCRD):

qPCR Data (CSV):

In the plots below, green is the positive control, blue are the samples, and red is the no template control (NTC).

Everything looks great! Nice, clean, gDNA-free RNA! Will proceed with reverse transcription.

Amplification Plots

Melt Curves

qPCR – Ronit’s DNAsed C.gigas Ploidy/Dessication RNA with 18s primers

After DNasing Ronit’s RNA earlier today, I needed to check for any residual gDNA.

Identified some old, old C.gigas 18s primers that should amplify gDNA:

  • gigas18s_fw (SRID 157)
  • gigas18s_rv (SRID 156)

Used some old C.gigas gDNA (BB15 from 20090519) as a positive control.

Samples were run on Roberts Lab CFX Connect (BioRad). All samples were run in duplicate. See qPCR Report (Results section) for plate layout, cycling params, etc.

qPCR master mix calcs (Google Sheet):

Results

qPCR Report (PDF):

qPCR File (PCRD):

qPCR Data (CSV):

Well, this primer set and/or the gDNA is not good. In the plots below, the positive control gNDA is in green, samples in blue, and no template controls (NTC) are in red.

Poor performance is most easily noticed when looking at the melt curves. They have multiple peaks, suggesting non-specific amplification, even in the positive control.

Additionally, although less evident from just looking at the plots, is the replicates are highly inconsistent. Although it’s possible that might be due to poor technique, it’s very unlikely.

Will have to identify different primers and/or positive control DNA.

Amplification Plots

Melt Curves

DNase Treatment – Ronit’s C.gigas Ploiyd/Dessication Ctenidia RNA

After quantifying Ronit’s RNA earlier today, I DNased them using the Turbo DNA-free Kit (Ambion), according to the manufacturer’s standard protocol.

Used 1000ng of RNA in a 50uL reaction in a 0.5mL thin-walled snap cap tube. Samples were mixed by finger flicking and then incubated 30mins @ 37oC in a PTC-200 thermal cylcer (MJ Research), without a heated lid.

DNase inactivation was performed (0.1 volumes of inactivation reagent; 5uL), pelleted, and supe transferred to new 1.7mL snap cap tube.

Samples were stored on ice in preparation for qPCR to test for residual gDNA.

DNase calculations are here:

Samples will be permanently stored here (Google Sheet):