Haynes Lab:Notebook/RNA-seq of PcTF Transfected U2OS & SK-N-SH cell lines/2015/01/22: 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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==Goals== | ==Goals== | ||
* | * Transfect U2OS cells | ||
* Induce KAH126/U2OS cells | |||
* purify KAH126/MV2 plasmid from E. coli | |||
<br><br> | <br><br> | ||
==Protocol== | ==Protocol== | ||
[http://openwetware.org/wiki/Haynes: | * [http://openwetware.org/wiki/Haynes:TransfectionPlasmid_Lipo mammalian tranfection w/lipofectamine] | ||
* Doxycycline induction of KAH126/U2OS cells - just add 1uL of doxycycline per mL of medium in each sample to be treated. | |||
* Plasmid prep - follow instructions in the Sigma plasmid mini-prep and in the cloning bootcamp | |||
<br><br> | <br><br> | ||
==Notes== | ==Notes== | ||
Observations, measurements, placeholder info etc. go here | Observations, measurements, placeholder info etc. go here. | ||
<br><br> | <br><br> | ||
==Results/Conclusions== | ==Results/Conclusions== | ||
Fluorescence microscopy images of KAH126/U2OS culture (taken today, induced on Monday) are below. | |||
[[Image:2015-01-22_Well_1_phase_%26_mCherry_10x.jpg | 500px | Well 1]] | |||
[[Image:2015-01-22_Well_2_phase_%26_mCherry_10x.jpg | 500px | Well 2]] | |||
[[Image:2015-01-22_Well_3_phase_%26_mCherry_10x.jpg | 500px | Well 3]] | |||
[[Image:2015-01-22_Well_4_phase_%26_mCherry_10x.jpg | 500px | Well 4]] | |||
[[Image:2015-01-22_Well_5_negative_control_phase_%26_mCherry_10x.jpg | 500px | Well 5 (negative control)]] | |||
[[Image:2015-01-22_Well_6_negative_control_phase_%26_mCherry_10x.jpg | 500px | Well 6 (negative control)]] | |||
----- | |||
DNA concentration data from plasmid mini-prep: | |||
{| {{table}} | |||
| align="center" style="background:#f0f0f0;"|'''Sample Read#''' | |||
| align="center" style="background:#f0f0f0;"|'''Location''' | |||
| align="center" style="background:#f0f0f0;"|'''260 Raw''' | |||
| align="center" style="background:#f0f0f0;"|'''280 Raw''' | |||
| align="center" style="background:#f0f0f0;"|'''320 Raw''' | |||
| align="center" style="background:#f0f0f0;"|'''260''' | |||
| align="center" style="background:#f0f0f0;"|'''280''' | |||
| align="center" style="background:#f0f0f0;"|'''260/280''' | |||
| align="center" style="background:#f0f0f0;"|'''ng/µL''' | |||
|- | |||
| 1||B2||0.157||0.108||0.058||0.097||0.048||2.017||97.095 | |||
|- | |||
| 1||B3||0.145||0.097||0.048||0.095||0.048||1.999||95.231 | |||
|- | |||
| 1||C2||0.12||0.089||0.056||0.061||0.031||1.937||60.704 | |||
|- | |||
| 1||C3||0.111||0.082||0.049||0.058||0.03||1.898||57.793 | |||
|} | |||
Average concentration of plasmid from colony 1: 96.163 ng/μL | |||
Average concentration of plasmid from colony 2: 59.2485 ng/μL | |||
NOTE: All of the plasmid collected from colony 1 was consumed in the transfection of the U2OS cells today. The wells transfected with the new plasmid were #'s 3 and 4. | |||
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Latest revision as of 00:40, 27 September 2017
RNA-seq of PcTF Transfected U2OS & SK-N-SH cell lines | Main project page Previous entry Next entry | |||||||||||||||||||||||||||||||||||||||||||||
SummaryToday the U2OS culture in 6-well plates looks ready for transfection (~80% confluency) and the KAH126/U2OS culture in 6-well plates looks ready for induction by doxycycline (~80% confluency). I'll be taking care of that, as well as running a plasmid mini-prep on some E. coli cells I grew up and harvested earlier in the week.
Goals
Protocol
NotesObservations, measurements, placeholder info etc. go here.
Results/ConclusionsFluorescence microscopy images of KAH126/U2OS culture (taken today, induced on Monday) are below. DNA concentration data from plasmid mini-prep:
Average concentration of plasmid from colony 1: 96.163 ng/μL Average concentration of plasmid from colony 2: 59.2485 ng/μL NOTE: All of the plasmid collected from colony 1 was consumed in the transfection of the U2OS cells today. The wells transfected with the new plasmid were #'s 3 and 4.
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