Haynes Lab:Notebook/RNA-seq of PcTF Transfected U2OS & SK-N-SH cell lines/2015/05/20: Difference between revisions
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|style="background-color: #EEE"|[[Image:owwnotebook_icon.png|128px]]<span style="font-size:22px;"> | |style="background-color: #EEE"|[[Image:owwnotebook_icon.png|128px]]<span style="font-size:22px;">Plasmid Maxiprep of pMaxGFP</span> | ||
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==Overview== | ==Overview== | ||
I'm making a lot of the pMaxGFP plasmid for use in transfection (Neon transfection, lipofectamine, etc.), as these processes require a very high concentration of very pure DNA. | |||
==Procedure== | |||
Yesterday I set up a 150mL culture of LB-Ampicillin from a DH5α-Turbo colony transformed with the pMaxGFP plasmid. I spun down the culture at 5000g for 10 minutes and discarded the supernatant, then followed the instructions for plasmid prep as laid out in the Sigma GenElute HP Endotoxin-Free Plasmid Maxiprep Kit. | |||
Plasmid concentration was measured on the Take3 Nanodrop. | |||
==DNA Quantification== | |||
Final volume of the purified plasmid is approximately 1.5 mL. | |||
{| {{table}} | |||
| align="center" style="background:#f0f0f0;"|'''Sample ID''' | |||
| align="center" style="background:#f0f0f0;"|'''Conc. (ng/µL)''' | |||
| align="center" style="background:#f0f0f0;"|'''St Dev (ng/µL)''' | |||
| align="center" style="background:#f0f0f0;"|'''260/280''' | |||
|- | |||
| pMaxGFP||486.333||7.966264997||1.888 | |||
|} | |||
I took 100µL of the purified plasmid and transferred it to a new sterile tube and am leaving that tube open in the BSC overnight to evaporate off and concentrate. Tomorrow I'll also perform a sodium acetate-ethanol precipitation with another aliquot of the plasmid. | |||
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Revision as of 15:32, 21 May 2015
 Plasmid Maxiprep of pMaxGFP
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OverviewI'm making a lot of the pMaxGFP plasmid for use in transfection (Neon transfection, lipofectamine, etc.), as these processes require a very high concentration of very pure DNA. ProcedureYesterday I set up a 150mL culture of LB-Ampicillin from a DH5α-Turbo colony transformed with the pMaxGFP plasmid. I spun down the culture at 5000g for 10 minutes and discarded the supernatant, then followed the instructions for plasmid prep as laid out in the Sigma GenElute HP Endotoxin-Free Plasmid Maxiprep Kit. Plasmid concentration was measured on the Take3 Nanodrop. DNA QuantificationFinal volume of the purified plasmid is approximately 1.5 mL.
I took 100µL of the purified plasmid and transferred it to a new sterile tube and am leaving that tube open in the BSC overnight to evaporate off and concentrate. Tomorrow I'll also perform a sodium acetate-ethanol precipitation with another aliquot of the plasmid.
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