Haynes Lab:Notebook/RNA-seq of PcTF Transfected U2OS & SK-N-SH cell lines/2015/01/29

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Owwnotebook icon.pngRNA-seq of PcTF Transfected U2OS & SK-N-SH cell lines Report.pngMain project page
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Summary

Today 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

  • SKNSH transfection with lipofectamine
  • Plasmid mini-prep
  • Plasmid restriction digestion
  • Run digestion products on agarose gel to check for purity



Protocol

Plasmid 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:

Reagent Volume
DNA(plasmid) 2.0 μL
10X buffer 1.5
EcoRI 1
PstI 1
dH2O 9.5
Total 15 μL

Incubate for 30min at 37°C, then run on a 1% agarose gel with 1kb ladder.



Notes

Observations, measurements, placeholder info etc. go here.



Results/Conclusions

Plasmid concentration:

' Colony 1 Colony 2
A260 0.0665 0.378
A280 0.033 0.196
260/280 1.9935 1.929
ng/uL 66.473 377.955

Gel image from plasmids purified over the last three weeks:

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.


SKNSH cells were transfected and placed back in the incubator. Tomorrow, check for transfection efficiency, and if >25% harvest on Monday.