Haynes Lab:Notebook/RNA-seq of PcTF Transfected U2OS & SK-N-SH cell lines/2015/01/08

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Summary

Following up on yesterday's work, today I'll be taking photographs of the transfected SKNSH cultures and checking for evidence of transfection, as shown by red fluorescence. If the samples show sufficiently high transfection efficiency, I'll move them into 10cm dishes to grow for 10 days, collecting samples at days 5 and 10 for RNA-seq and flow cytometry.

I'm also continuing to grow the DH5α cells for harvesting of plasmid.



Goals

  • Photograph SKNSH cells transfected with PcTF-RFP plasmid using phase contrast & red fluorescence
  • Split transfected cells (and control) into 2 dishes per sample for 5 and 10 day sampling
  • Continue with plasmid harvesting



Protocol

passaging adherent cells
Cloning Bootcamp, Session 3 - Plasmid Extraction and Validation



Materials

  • List materials here, to be gathered before the experiment
  • Consumables such as media, antibiotics, DNA, kits etc.



Notes

DH5α KAH126/MV2 colonies

Growth of DH5α containing PcTF-RFP expressing plasmid was a success. Both liquid colonies grew as well (not shown).



Results/Conclusions

Plasmid concentration for KAH126/MV2:

2 3
A 260
280
260/280
ng/µL
Sample 1 0.038 260
0.021 280
1.802 260/280
37.774 ng/µL
Sample 2 0.031 260
0.018 280
1.756 260/280
31.39 ng/µL

Yield is fairly low, at 38 ng/μL and 31 ng/μL for samples 1 and 2, respectively. Samples have been placed in the -20 freezer in the main lab, in Ben's Box.

SKNSH samples transfected with PcTF-RFP and one negative control well have each been split into two 10cm cell culture dishes, for a total of 8 samples. The first round of collection will be on Day 5 post-transfection, which is Monday January 12th. The second round of collection will be on Day 10, Saturday January 17th.