--- author: Sam White toc-title: Contents toc-depth: 5 toc-location: left layout: post title: qPCR - P.generosa RPL5-v2-v3 and TIF3s6b-v2-v3 Primer Tests date: '2020-08-24 09:52' tags: - qPCR - CFX Connect - Panopea generosa - geoduck - SsoFast categories: - 2020 - Miscellaneous --- Shelly ordered some new primers as potential normalizing genes [and asked me to test them out](https://github.com/RobertsLab/resources/issues/988) (GitHub Issue). Primers used: | SRID | Primer_Name | |------|----------------| | 1787 | RPL5_v2_FWD | | 1786 | RPL5_v2_REV | | 1785 | RPL5_v3_FWD | | 1784 | RPL5_v3_REV | | 1783 | TIF3s6b_v2_FWD | | 1782 | TIF3s6b_v2_REV | | 1781 | TIF3s6b_v3_FWD | | 1780 | TIF3s6b_v3_REV | Positive control was pooled cDNA, created by combining 2uL from each of the following: - 11-08 1H ([made by me from 20191125](https://robertslab.github.io/sams-notebook/posts/2019/2019-11-26-Reverse-Transcription---P.generosa-DNased-Hemolypmh-and-Hemocyte-RNA-from-20191125/)) - 11-08 2H ([made by me from 20191125](https://robertslab.github.io/sams-notebook/posts/2019/2019-11-26-Reverse-Transcription---P.generosa-DNased-Hemolypmh-and-Hemocyte-RNA-from-20191125/)) - 57H ([made by me from 20191125](https://robertslab.github.io/sams-notebook/posts/2019/2019-11-26-Reverse-Transcription---P.generosa-DNased-Hemolypmh-and-Hemocyte-RNA-from-20191125/)) - 11/15 Chew (made by Kaitlyn, no date on tube) - 11/21 Star (made by Kaitlyn, no date on tube) I also used [geoduck gDNA (162ng/uL; from 20170105)](https://robertslab.github.io/sams-notebook/posts/2017/2017-01-05-dna-isolation-geoduck-gdna-for-illumina-initiated-sequencing-project/) as a potential positive control, and/or as confirmation that these primers will/not amplify gDNA. Master mix calcs are here: - [200200824_qPCR_geoduck_RPL5-v2-v3_TIF2s6b-v2-v3](https://docs.google.com/spreadsheets/d/1nA4_esJW8ex81fQXtI8KLdZKqDPWBFrRFoRbXU0Wfqw/edit?usp=sharing) (Google Sheet) All qPCR reactions were run in duplicate. See qPCR Report (Results section below) for plate layout, cycling params, etc. --- # RESULTS qPCR Report (PDF): - [sam_2020-08-24_05-17-26_BR006896.pdf](https://owl.fish.washington.edu/Athaliana/qPCR_data/qPCR_reports/sam_2020-08-24_05-17-26_BR006896.pdf) CFX Data File (PCRD): - [sam_2020-08-24%2005-17-26_BR006896.pcrd](https://owl.fish.washington.edu/scaphapoda/qPCR_data/cfx_connect_data/sam_2020-08-24%2005-17-26_BR006896.pcrd) CFX Results File (CSV): - [sam_2020-08-24_05-17-26_BR006896_Quantification-Cq-Results.csv](https://owl.fish.washington.edu/Athaliana/qPCR_data/sam_2020-08-24_05-17-26_BR006896_Quantification-Cq-Results.csv) All the primers look good: - Cq is reasonably low - Melt curves have single peak Amplifcation/melt plots for each primer are below. NOTE: Genomic DNA is amplified by all three primer sets and comes up at an earlier Cq than cDNA. In `TIF3s6b v3`, gDNA exhibits a small, secondary peak at ~75oC. ##### RPL5 v2 AMPLIFICATION PLOTS ![RPL5 v2 amplification plots](https://owl.fish.washington.edu/Athaliana/qPCR_data/sam_2020-08-24_05-17-26_BR006896_RPL5-v2_amp_plots.png) MELT PLOTS ![RPL5 v2 melt plots](https://owl.fish.washington.edu/Athaliana/qPCR_data/sam_2020-08-24_05-17-26_BR006896_RPL5-v2_melt_plots.png) ##### RPL5 v3 ![RPL5 v3 amplification plots](https://owl.fish.washington.edu/Athaliana/qPCR_data/sam_2020-08-24_05-17-26_BR006896_RPL5-v3_amp_plots.png) ![RPL5 v3 melt plots](https://owl.fish.washington.edu/Athaliana/qPCR_data/sam_2020-08-24_05-17-26_BR006896_RPL5-v3_melt_plots.png) ##### TIF3s6b v2 ![TIF3s6b v2 amplification plots](https://owl.fish.washington.edu/Athaliana/qPCR_data/sam_2020-08-24_05-17-26_BR006896_TIF3s6b-v2_amp_plots.png) ![TIF3s6b v2 melt plots](https://owl.fish.washington.edu/Athaliana/qPCR_data/sam_2020-08-24_05-17-26_BR006896_TIF3s6b-v2_melt_plots.png) ##### TIF3s6b v3 ![TIF3s6b v3 amplification plots](https://owl.fish.washington.edu/Athaliana/qPCR_data/sam_2020-08-24_05-17-26_BR006896_TIF3s6b-v3_amp_plots.png) ![TIF3s6b v3 melt plots](https://owl.fish.washington.edu/Athaliana/qPCR_data/sam_2020-08-24_05-17-26_BR006896_TIF3s6b-v3_melt_plots.png)