Haynes Lab:Notebook/Engineering PC-TFs/2013/09/26: Difference between revisions

From OpenWetWare
Jump to navigationJump to search
Line 46: Line 46:
|}
|}


* Follow steps for gel electrophoresis.
*  Orient top notch of gel electrophoresis apparatus. Place gel tray into larger tray.
*  Fill gel flask with up to 60 ml of TA buffer.
*  Create 1% gel by putting .6 grams of agarose into flask.
*  Microwave agarose solution for 40 seconds
*  Using hot gloves, grab flask out of microwave and swirl flask vigorously. Place back in microwave for 40 more seconds.
*  When flask is taken out of microwave, make sure that the agarose is completed dissolved.
*  Set up wells on gel electrophoresis apparatus. The mold for the first digest is B-10. Use the thick "teeth" and slip in the notches.
*  Add EtBr (ethidium bromide) to the agarose gel. There should be 1 microliter of EtBr per 10ml of agarose gel. Therefore, 6 µL.
*  Pour gel into tray.
*  Wash the agarose gel flask.
* Run gel for 45 min at 100 V. Check gel under UV light.




Revision as of 19:30, 26 September 2013



 		<html><img src="/images/9/94/Report.png" border="0" /></html> Main project page
<html><img src="/images/c/c3/Resultset_previous.png" border="0" /></html>Previous entry<html>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</html>Next entry<html><img src="/images/5/5c/Resultset_next.png" border="0" /></html>

Summary

  • Perform restriction digest for CMV promoter
Reagent Volume
DNA (plasmid) 10 μL
XbaI 1.0 μL
PstI 1.0 μL
Green 10x FastDigest buffer 3.0 μL
dH2O 15 μL
  30 μL --> 37°C/ ~10 min.


  • Measure the following into 1.5 mL tubes.
Reagent 15% 10% 7% 5% 2%
30% PEG 50 μL 33 μL 23 μL 17 μL 7 μL
DNA 15μL 15 μL 15 μL 15 μL 15 μL
dH2O 35 μL 52 μL 62 μL 68 μL 78 μL
Total 100 μL 100 μL 100 μL 100 μL 100 μL
  • Follow steps for gel electrophoresis.
  • Orient top notch of gel electrophoresis apparatus. Place gel tray into larger tray.
  • Fill gel flask with up to 60 ml of TA buffer.
  • Create 1% gel by putting .6 grams of agarose into flask.
  • Microwave agarose solution for 40 seconds
  • Using hot gloves, grab flask out of microwave and swirl flask vigorously. Place back in microwave for 40 more seconds.
  • When flask is taken out of microwave, make sure that the agarose is completed dissolved.
  • Set up wells on gel electrophoresis apparatus. The mold for the first digest is B-10. Use the thick "teeth" and slip in the notches.
  • Add EtBr (ethidium bromide) to the agarose gel. There should be 1 microliter of EtBr per 10ml of agarose gel. Therefore, 6 µL.
  • Pour gel into tray.
  • Wash the agarose gel flask.
  • Run gel for 45 min at 100 V. Check gel under UV light.



Retrieved from "https://openwetware.org/mediawiki/index.php?title=Haynes_Lab:Notebook/Engineering_PC-TFs/2013/09/26&oldid=732387"