Haynes Lab:Notebook/RNA-seq of PcTF Transfected U2OS & SK-N-SH cell lines/2015/01/29: Difference between revisions
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|style="background-color: #EEE"|[[Image:owwnotebook_icon.png|128px]]<span style="font-size:22px;">RNA-seq of PcTF Transfected U2OS & SK-N-SH cell lines</span> | |style="background-color: #EEE"|[[Image:owwnotebook_icon.png|128px]]<span style="font-size:22px;">RNA-seq of PcTF Transfected U2OS & SK-N-SH cell lines</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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For reference on plasmid restriction digestion and expected size, see Karmella's notes [http://openwetware.org/wiki/User:Karmella_Haynes/Notebook/BioBrick_cloning/2009/12/16 here]. | |||
Restriction digestion setup: | Restriction digestion setup: | ||
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Gel image from plasmids purified over the last three weeks: | Gel image from plasmids purified over the last three weeks: | ||
[[Image:2015-01-29_Ben_KAH126-MV2_digestion.JPG | 500px | EcoRI & PstI digestion of KAH126-MV2 plasmid]] | |||
Expected size fragments are 4.5kb for MV2 backbone, 1.9kb for PcTF-RFP insert | |||
Samples from left to right: | |||
Ladder, 31ng/μL, 38ng/μL, empty, 61ng/μL, empty, 66ng/μL, 378ng/μL, ladder, negative control | |||
All plasmids collected look fairly clean, though only one is of high enough concentration to use in transfection. | |||
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Latest revision as of 00:41, 27 September 2017
 RNA-seq of PcTF Transfected U2OS & SK-N-SH cell lines
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SummaryToday I'll be performing another transfection of SKNSH samples, as well as purifying plasmid and running it on a gel to check for purity.
Goals
ProtocolPlasmid mini-prep: Follow instructions in kit transfection with lipofectamine For reference on plasmid restriction digestion and expected size, see Karmella's notes here. Restriction digestion setup:
Incubate for 30min at 37°C, then run on a 1% agarose gel with 1kb ladder.
NotesObservations, measurements, placeholder info etc. go here.
Results/ConclusionsPlasmid concentration:
Gel image from plasmids purified over the last three weeks:
Expected size fragments are 4.5kb for MV2 backbone, 1.9kb for PcTF-RFP insert Samples from left to right: Ladder, 31ng/μL, 38ng/μL, empty, 61ng/μL, empty, 66ng/μL, 378ng/μL, ladder, negative control All plasmids collected look fairly clean, though only one is of high enough concentration to use in transfection. SKNSH cells were transfected and placed back in the incubator. Tomorrow, check for transfection efficiency, and if >25% harvest on Monday.
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