Haynes Lab:Notebook/RNA-seq of PcTF Transfected U2OS & SK-N-SH cell lines/2015/06/03: Difference between revisions
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|style="background-color: #EEE"|[[Image:owwnotebook_icon.png|128px]]<span style="font-size:22px;">K562 Growth and Experimental Design</span> | |style="background-color: #EEE"|[[Image:owwnotebook_icon.png|128px]]<span style="font-size:22px;">K562 Growth and Experimental Design</span> | ||
|style="background-color: #F2F2F2" align="center"| | |style="background-color: #F2F2F2" align="center"|[[File:Report.png|frameless|link={{#sub:{{FULLPAGENAME}}|0|-11}}]][[{{#sub:{{FULLPAGENAME}}|0|-11}}|Main project page]]<br />{{#if:{{#lnpreventry:{{FULLPAGENAME}}}}|[[File:Resultset_previous.png|frameless|link={{#lnpreventry:{{FULLPAGENAME}}}}]][[{{#lnpreventry:{{FULLPAGENAME}}}}{{!}}Previous entry]] }}{{#if:{{#lnnextentry:{{FULLPAGENAME}}}}|[[{{#lnnextentry:{{FULLPAGENAME}}}}{{!}}Next entry]][[File:Resultset_next.png|frameless|link={{#lnnextentry:{{FULLPAGENAME}}}}]]}} | ||
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==Experimental Design== | ==Experimental Design== | ||
The plan is to collect 1 control (GFP) and 2 samples (KAH126-MV2) for each day of collection, and to collect on days 2 and 3. This makes 6 samples total. I'll be using a 6-well plate for this experiment and transfecting using the 100 µL tips. | The plan is to collect 1 control (GFP) and 2 samples (KAH126-MV2) for each day of collection, and to collect on days 2 and 3 (24 hours & 48 hours post-transfection). This makes 6 samples total. I'll be using a 6-well plate for this experiment and transfecting using the 100 µL tips. | ||
Cells will be resuspended in buffer at 2.0 x 10<sup>7</sup> cells/mL for transfection. Multiplied by 8 transfections (2 buffer), that's 2.0E7 cells/mL * 0.8 mL = 1.6E7 cells needed. | Cells will be resuspended in buffer at 2.0 x 10<sup>7</sup> cells/mL for transfection. Multiplied by 8 transfections (2 buffer), that's 2.0E7 cells/mL * 0.8 mL = 1.6E7 cells needed. |
Latest revision as of 01:00, 27 September 2017
K562 Growth and Experimental Design | Main project page Previous entry Next entry |
OverviewOn Monday I started a 20 mL culture of K562 in antibiotic-free medium. Today I'll be checking cell density and planning out the transfection setup. Cell DensityMeasured at 300,000 cells/mL by hemacytometer. 3.0E5 cells/mL * 20 mL = 6.0E6 cells total Experimental DesignThe plan is to collect 1 control (GFP) and 2 samples (KAH126-MV2) for each day of collection, and to collect on days 2 and 3 (24 hours & 48 hours post-transfection). This makes 6 samples total. I'll be using a 6-well plate for this experiment and transfecting using the 100 µL tips. Cells will be resuspended in buffer at 2.0 x 107 cells/mL for transfection. Multiplied by 8 transfections (2 buffer), that's 2.0E7 cells/mL * 0.8 mL = 1.6E7 cells needed. Number of cells per well: 0.4-1 x 106 Initial cell density after transfection and transferring to wells (in 2 mL plating medium) will be 2.0E7 cells/mL * 0.1 mL / 2.0 mL = 1.0E6 cells/mL Amount of DNA per sample is 5-30 µg, not to exceed 10% of total volume of cells (100 µL * 0.1 = 10 µL). Both pMaxGFP and KAH126-MV2 are at concentrations of 2.0 µg/µL, so 10 µL per sample would be 20 µg of DNA per sample. Procedure
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