Difference between revisions of "Haynes Lab:Notebook/Engineering PC-TFs/2013/03/03"

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(Summary)
(Summary)
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==Summary==
 
==Summary==
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<font size=3>''E/P Digest for FURI 2013-2014 application''</font>
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* Retrieve BL01, BL05, BL09, BL011 assembly from freezer and thaw at room temperature.
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* Also let restriction enzymes thaw.
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*Do restriction digest (EcoRI/PstI)''Diagnostic digest''
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{| {{table}}
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| align="center" style="background:#f0f0f0;"|
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| align="center" style="background:#f0f0f0;"|'''15 µl Total'''
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| align="center" style="background:#f0f0f0;"|'''Master Mix'''
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|-
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| ||1 rxn||x 4
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|-
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| DNA plasmid||3||--
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|-
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| enzyme 1||1||4
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|-
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| enzyme 2||1||4
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|-
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| 10x buffer||1.5||6
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|-
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| dH2O||8.5||34
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|-
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|
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|}
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*Aliquot 12 μL to each tube. Then add 3 μL DNA to each tube.
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*Place each tube in heat block 37°C.
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Follow steps for gel electrophoresis.
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*  Orient top notch of gel electrophoresis apparatus. Place gel tray into larger tray.
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*  Fill gel flask with up to 60 ml of TA buffer.
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*  Create 1% gel by putting .6 grams of agarose into flask.
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*  Microwave agarose solution for 40 seconds
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*  Using hot gloves, grab flask out of microwave and swirl flask vigorously. Place back in microwave for 40 more seconds.
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*  When flask is taken out of microwave, make sure that the agarose is completed dissolved.
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*  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.
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*  Add EtBr (ethidium bromide) to the agarose gel. There should be 1 microliter of EtBr per 10ml of agarose gel. Therefore, 6 µL.
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*  Pour gel into tray.
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*  Wash the agarose gel flask.
 +
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* Run gel for 30 min at 100 V. Check gel under UV light.
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* Label Tubes
 
* Label Tubes

Revision as of 21:43, 3 March 2013

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Summary

E/P Digest for FURI 2013-2014 application

  • Retrieve BL01, BL05, BL09, BL011 assembly from freezer and thaw at room temperature.
  • Also let restriction enzymes thaw.
  • Do restriction digest (EcoRI/PstI)Diagnostic digest
15 µl Total Master Mix
1 rxn x 4
DNA plasmid 3 --
enzyme 1 1 4
enzyme 2 1 4
10x buffer 1.5 6
dH2O 8.5 34
  • Aliquot 12 μL to each tube. Then add 3 μL DNA to each tube.
  • Place each tube in heat block 37°C.

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 30 min at 100 V. Check gel under UV light.


  • Label Tubes

1. BL01 2. BL05 3. BL09

  • 2 mL LB Broth + amp added to test tubes
  • Use pipette tips and dip in bacteria colony.