Haynes Lab:Notebook/Engineering PC-TFs/2013/04/21

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(Summary)
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==Summary==
==Summary==
-
*
 
 +
* Cutting inserts to be placed in mammalian vector
 +
A) Get biobricks and enzymes from freezer.
 +
 +
PstI, SpeI
 +
 +
The following list of parts are:
 +
* hPCD
 +
* fshPCD
 +
* flyPCD
 +
 +
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.
 +
 +
{| {{table}} cellspacing="3" <!-- Digest rxn. table -->
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|- valign="top"
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| bgcolor=#cfcfcf | '''Reagent'''
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| bgcolor=#cfcfcf | '''Volume'''
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| rowspan="7" |  <br>30 μL/lane, 1% agarose; [http://openwetware.org/wiki/Image:KAH_Fermentas_GeneRuler_1kbplus.jpg Ladder]
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|-
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| DNA (plasmid) || 20.0 μL
 +
|-
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| SpeI|| 1.0 μL
 +
|-
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| PstI|| 1.0 μL
 +
|-
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| Green 10x FastDigest buffer || 3.0 μL
 +
|-
 +
| dH<sub>2</sub>O || 5 μL
 +
|-
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| &nbsp; || 30 μL --> 37°C/ ~10 min.
 +
|}
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Revision as of 02:12, 23 April 2013

Engineering PC-TFs Main project page
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Summary

  • Cutting inserts to be placed in mammalian vector

A) Get biobricks and enzymes from freezer.

PstI, SpeI

The following list of parts are:

  • hPCD
  • fshPCD
  • flyPCD

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.
Reagent Volume
30 μL/lane, 1% agarose; Ladder
DNA (plasmid) 20.0 μL
SpeI 1.0 μL
PstI 1.0 μL
Green 10x FastDigest buffer 3.0 μL
dH2O 5 μL
  30 μL --> 37°C/ ~10 min.


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