Haynes Lab:Notebook/Engineering PC-TFs/2013/03/18

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==Summary==
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==3/18/22==
Cutting inserts to be placed in mammalian vector
Cutting inserts to be placed in mammalian vector
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* '''Procedure'''
 
''' A) Get biobricks and enzymes from freezer.'''
''' A) Get biobricks and enzymes from freezer.'''
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SpeI, PstI, XbaI
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PstI, XbaI
The following list of parts are:
The following list of parts are:
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* BL01  
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* 1. BL01  
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* BL05  
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* 2. BL05  
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* BL09
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* 3. BL09
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* BL12
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* 4. BL12
{| {{table}}
{| {{table}}
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<font size=3>''B)Follow digest procedure:''
<font size=3>''B)Follow digest procedure:''
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  DNA          20.0 µL
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*DNA          20.0 µL
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  Enzyme 1    1.0  µL
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*Enzyme 1    1.0  µL
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  Enzyme 2    1.0  µL
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*Enzyme 2    1.0  µL
-
  10x buffer  3.0  µL
+
*10x buffer  3.0  µL
-
  dH2O        5.0  uL
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*dH2O        5.0  uL          
-
           
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*            = 30.0 uL </font>  
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              = 30.0 uL </font>  
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|}
|}
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''Digest Order:''
''Digest Order:''
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*4  BL12      XbaI/PstI
*4  BL12      XbaI/PstI
 +
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.
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* Run gel for 45 min at 100 V. Check gel under UV light.
{| {{table}} cellspacing="3" <!-- Digest rxn. table -->
{| {{table}} cellspacing="3" <!-- Digest rxn. table -->
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| PstI|| 1.0 μL
| PstI|| 1.0 μL
|-
|-
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|10x FastDigest buffer + green loading dye  || 1.0 μL
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| Green 10x FastDigest buffer || 1.0 μL
|-
|-
| dH<sub>2</sub>O || 10 μL
| dH<sub>2</sub>O || 10 μL

Revision as of 22:34, 22 March 2013

Engineering PC-TFs Main project page
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3/18/22

Cutting inserts to be placed in mammalian vector

A) Get biobricks and enzymes from freezer.

PstI, XbaI

The following list of parts are:

  • 1. BL01
  • 2. BL05
  • 3. BL09
  • 4. BL12
Stock Digest

B)Follow digest procedure:

  • DNA 20.0 µL
  • Enzyme 1 1.0 µL
  • Enzyme 2 1.0 µL
  • 10x buffer 3.0 µL
  • dH2O 5.0 uL
  • = 30.0 uL

Digest Order:

Biobrick Restriction Enzymes

  • 1. BL01 XbaI/PstI
  • 2. BL05 XbaI/PstI
  • 3. BL09 XbaI/PstI
  • 4 BL12 XbaI/PstI

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 Stock digest 3/18/2013 Stock digest 3/18/2013
30 μL/lane, 1% agarose; Ladder
DNA (plasmid) 15.0 μL
XbaI 1.0 μL
PstI 1.0 μL
Green 10x FastDigest buffer 1.0 μL
dH2O 10 μL
  30 μL --> 37°C/ ~10 min.


# Biobrick ng/µL
1 hPCD : mCh : SP1AB (BL01) 18.834
2 fshPCD : mCh : SP1AB (BL05) 14.471
3 flyPCD : mCh : SP1A (BL09) 7.302
4 hPCD : BL01 (BL12) 10.84



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