Tag Archives: actin

qPCR – Jake’s O.lurida ctenidia 1hr post-mechanical stress DNased RNA

Ran qPCR on DNased RNA from earlier today to assess whether there was any residual gDNA after the DNase treatment with Oly_Actin_F/R primers (SR IDs: 1505, 1504).

Used 1μL from all templates.

All samples were run in duplicate.

Positive control was HL1 O.lurida DNA isolated by Jake on 20150323.

Cycling params:

  • 95C – 2.5mins
  • 40 cycles of:
    • 95C – 10s
    • 60C – 20s
  • Melt curve

Master mix calcs are here: 201500806_qPCR_Oly_DNased_RNA

qPCR Plate Layout: 20150806_qPCR_plate_Jake_Oly_DNased_RNA

Results:

qPCR Data File (Opticon): 20150806_165044.tad
qPCR Report (Google Spreadsheet): 20150806_qPCR_Report_Jake_Oly_DNased_RNA

Positive control comes up around cycle ~21.

No amplification in the no template controls.

Two wells of the DNased RNA samples exhibit amplification (E3, F6), however the corresponding respective replicate does not. Will proceed with reverse transcription.

 

Amplification Plots

Positive Controls

 

Melt Curves

Positive Controls (HL1)

DNased RNA Samples

Follow the green and red lines with the vertical bars. The different colors reflect that those are two different samples. Additionally, their respective replicates do not exhibit amplification.

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qPCR – Re-run Jake’s O.lurida DNased RNA Samples NC1, SC1, SC2, SC4 from 20150514

The following DNased RNA samples showed inconsistencies between qPCR reps (one rep showed amplification, the other rep did not) on 20150514:

  • NC1
  • SC1
  • SC2
  • SC4

Reran these four samples to obtain a definitive answer as to whether or not they have residual gDNA in them prior to using them to make cDNA.

Used Oly_Actin primers (SR IDs: 1504, 1505)

Used 1μL from all templates.

All samples were run in duplicate.

Positive control was HL1 O.lurida DNA isolated by Jake on 20150323.

Cycling params:

  • 95C – 2.5mins
  • 40 cycles of:
    • 95C – 10s
    • 60C – 20s
  • Melt curve

Master mix calcs: 20150521_qPCR_Oly_DNased_RNA

Plate layout: 20150521_qPCR_plate_Jake_Oly_DNased_RNA

Results:

qPCR Data File (Opticon): Sam_20150521_145749.tad
qPCR Report (Google Sheet): 20150521_qPCR_Report_Jake_Oly_DNased_RNA

 

No amplification in any of the RNA samples, nor the NTCs. Will make cDNA.

 

Amplification Plots

 

 

Melt Curves

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qPCR – Jake’s O.lurida ctenidia DNased RNA (1hr Heat Shock Samples)

Ran qPCR on DNased RNA from earlier today to assess whether there was any residual gDNA after the DNase treatment with Oly_Actin_F/R primers (SR IDs: 1505, 1504).

Used 1μL from all templates.

All samples were run in duplicate.

Positive control was HL1 O.lurida DNA isolated by Jake on 20150323.

Cycling params:

  • 95C – 2.5mins
  • 40 cycles of:
    • 95C – 10s
    • 60C – 20s
  • Melt curve

Master mix calcs are here (used same calcs from the other day): 20150512_qPCR_Oly_RNA

Plate layout: 20150514_qPCR_plate_Jake_Oly_1hr_HS_DNased_RNA

Results:

qPCR Data File (Opticon): Sam_20150514_170332.tad

qPCR Report (Google Spreadsheeet): 20150514_qPCR_Report_Jake_Oly_DNased_1hr_HS_RNA

 

Positive control samples are the only samples that produced amplification (cycle ~20). Will proceed to making cDNA.

 

Amplification Plots

 

Melt Curves

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qPCR – Jake’s O.lurida ctenidia DNased RNA (Control Samples)

Ran qPCR on DNased RNA from earlier today to assess whether there was any residual gDNA after the DNase treatment with Oly_Actin_F/R primers (SR IDs: 1505, 1504).

Used 1μL from all templates.

All samples were run in duplicate.

Positive control was HL1 O.lurida DNA isolated by Jake on 20150323.

Cycling params:

  • 95C – 2.5mins
  • 40 cycles of:
    • 95C – 10s
    • 60C – 20s
  • Melt curve

Master mix calcs are here: 20150514_qPCR_Oly_DNased_RNA

qPCR Plate Layout: 20150514_qPCR_plate_Jake_Oly_Control_RNA

Results:

qPCR Data File (Opticon): Sam_20150514_153529.tad

qPCR Report (Google Spreadsheet): 20150514_qPCR_Report_Jake_Oly_DNased_Control_RNA

Positive control comes up around cycle ~21.

No amplification in the no template controls.

Four wells of the DNased RNA samples exhibit amplification (B5, C10, C12, D3), however each respective replicate does not. Will re-test these four samples (NC1, SC1, SC2, SC4).

 

Amplification Plots

 

Melt Curves

 

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qPCR – Jake O.lurida ctenidia RNA (Heat Shock Samples) from 20150506

Ran qPCRs on the O.lurida total RNA I isolated on 20150506 to assess presence of gDNA carryover with Oly Actin primers (SR IDs: 1505, 1504).

Used 1μL from all templates.

All samples were run in duplicate.

Positive control was HL1 O.lurida DNA isolated by Jake on 20150323.

Master mix calcs are here: 20150512_qPCR_Oly_RNA

Cycling params:

  • 95C – 3mins
  • 40 cycles of:
    • 95C – 5s
    • 60C – 20s
  • Melt curve

 

Plate layout: 20150512_qPCR_plate_Jake_Oly_HS_RNA

Results:

qPCR Data File (Opticon2): Sam_20150512_123246.tad

qPCR Report (Google Spreadsheet):20150512_qPCR_Report_Jake_Oly_HS_RNA

Excluding the no template controls (NTC), all samples produced amplification. Will require DNasing before making cDNA.

Related to the qPCR I ran earlier today with these same primers, the efficiencies of the reactions on this plate are significantly better (i.e. normal; >80% efficiencies) than the earlier qPCR. The improved efficiency would also explain why the positive control comes up two cycles earlier on this run.

In the amplification plots below, the positive control reps are the two lines coming up at cycle ~20.

 

Amplification Plots

 

Melt Curves

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qPCR – Jake O.lurida ctenidia RNA (Control Samples) From 20150507

Ran qPCRs on the O.lurida total RNA I isolated on 20150507 to assess presence of gDNA carryover with Oly Actin primers (SR IDs: 1505, 1504).

Used 1μL from all templates.

All samples were run in duplicate.

Positive control was HL1 O.lurida DNA isolated by Jake on 20150323.

Master mix calcs are here: 20150512_qPCR_Oly_RNA

Cycling params:

  • 95C – 3mins

40 cycles of:

  • 95C – 5s
  • 60C – 20s

Melt curve

 

Plate layout: 20150512_qPCR_plate_Jake_Oly_Control_RNA

 

Results:

qPCR Data File (Opticon2): Sam_20150512_105811.tad

qPCR Report (Google Spreadsheet): 20150512_qPCR_Report_Jake_Oly_Control_RNA

Excluding the no template controls (NTC), all samples produced amplification. Will require DNasing before making cDNA.

On a side note, it should be noted that the efficiencies for all of the reactions were pretty bad; probably averaging 50%. Not entirely sure why or what that indicates.

In the amplification plots below, the positive control reps are the two red lines coming up at cycle ~22.

Amplification Plots

 

 

Melt Curves

 

 

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qPCR – C.gigas actin and GAPDH on V.vulnificus exposure cDNA (from 20110311)

Ran a qPCR on all cDNA samples from the V.vulnificus exposure experiment from 20110111. This qPCR was to test 2 of 4 potential normalizing genes to evaluate which genes show the least amount of effect from the treatments in this experiment. Primers for actin used were Cg_Actin_306_F (SR ID: 1170), Cg_Actin_408_R (SR ID: 1171). Samples were run in duplicate. Master mix calcs are here. The master mix info is the same that was used earlier today, but with the primers noted above, not those listed on the calcs page. Plate layout, cycling params, etc., can be seen in the qPCR Report (see Results).

Results:

qPCR Data File (BioRad CFX96)

qPCR Report (PDF)

Actin: Average Cq = 20.21, Standard Deviation = 1.22

GAPDH: Average Cq = 24.42, Standard Deviation = 0.519

Based on the results from the 4 normalizing genes examined, I will use GAPDH as the normalizing gene due to it having the lowest standard deviation of the 4 normalizing genes. Will perform another qPCR to run a duplicate of GAPDH so that we have a second rep.

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PCR – Bay/Sea Scallop DNA

An additional attempt to get the actin primers to work for use in screening samples for bay/sea scallop hybrids. The scallop_actin_fw primer was used in conjunction with the following:

bay_actin_Rv0 (Rxn 1)

bay_actin_Rv2 (Rxn 2)

sea_actin_Rv4 (Rxn 3)

sea_actin_Rv5 (Rxn 4)

PCR set up is here. Just used Bay or Sea scallop gDNA (chelexed). When/If get this working correctly, will start screening the hybrid samples. Anneal of 53C.

Lane 1 – 100bp Ladder

Lane 2 – Rxn 1: Bay

Lane 3 – Rxn 1: Sea

Lane 4 – Rxn 1: H2O

Lane 5 – Rxn 1: H2O

Lane 6 – Rxn 2: Bay

Lane 7 – Rxn 2: Sea

Lane 8 – Rxn 2: H2O

Lane 9 – Rxn 2: H2O

Lane 10 – Rxn 3: Bay

Lane 11 – Rxn 3: Sea

Lane 12 – Rxn 3: H2O

Lane 13 – Rxn 3: H2O

Lane 14 – Rxn 4: Bay

Lane 15 – Rxn 4: Sea

Lane 16 – Rxn 4: H2O

Lane 17 – Rxn 4: H2O

Lane 18 – 100bp Ladder

Results: Rxn 1 shows amplification with both Bay & Sea Scallop gDNA. The bands are close in size, but look like they would be more distinguishable if run on higher percentage gel and for a longer period of time to get better separation. However, there is contamination in one of the two water samples..

Rxn 2 shows amplification of only the Bay Scallop gDNA.

Rxn 3 shows amplification in both Bay & Sea Scallop gDNA and both bands are of the exact same size.

Rxn 4 shows no amplification in either set of gDNA.

Using the primers used in Rxn 1 will probably allows us to succesfully screen potential hybrids. Just need to remember to use high-percentage agarose gels and run samples for longer periods of time to get sufficient separation.

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PCR – Bay/Sea scallop gDNAs

Used higher annealing temps to improve primer specificity, compared to yesterday’s results. PCR set up and plate layout is here.

See the PCR/plate set up link for samples. Hyperladder is placed between every 12 samples.

Results: See this Google Spreadsheet for a summary of the 4 gels from the last two days.

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PCR – Bay/Sea scallop gDNA isolated earlier today

Used 3 sets of reverse primers:

Bay_Actin_Rv0

Bay_Actin_Rv2

Sea_Actin_Rv2

Primers were slected based on information from Steven’s notebook (#8, 12/30/2007-1/3/2008). Anneal temp 53C.

PCR set up here . Plate layout here .

Samples were run out by Steven the following day.

Gel 1 of 3

Gel 2 of 3

Gel 3 of 3

Results:

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