Haynes Lab:Notebook/RNA-seq of PcTF Transfected U2OS & SK-N-SH cell lines/2015/01/22

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Summary

Today 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

  • Transfect U2OS cells
  • Induce KAH126/U2OS cells
  • purify KAH126/MV2 plasmid from E. coli



Protocol

  • 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




Notes

Observations, measurements, placeholder info etc. go here.



Results/Conclusions

Fluorescence microscopy images of KAH126/U2OS culture (taken today, induced on Monday) are below.

Well 1

Well 2

Well 3

Well 4

Well 5 (negative control)

Well 6 (negative control)


DNA concentration data from plasmid mini-prep:

Sample Read# Location 260 Raw 280 Raw 320 Raw 260 280 260/280 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.