Haynes Lab:Notebook/Engineering PC-TFs/2013/03/25

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(Summary)
Line 11: Line 11:
''' A) Get biobricks and enzymes from freezer.'''
''' A) Get biobricks and enzymes from freezer.'''
-
PstI, XbaI
+
PstI, SpeI
The following list of parts are:
The following list of parts are:
-
* 1. BL01
+
* Kozak
-
* 2. BL05
+
-
* 3. BL09
+
-
* 4. BL12
+
-
 
+
{| {{table}}
{| {{table}}
|align="center" style="background:#f0f0f1;"|'''Stock Digest'''
|align="center" style="background:#f0f0f1;"|'''Stock Digest'''
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|}
|}
-
''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.
Follow steps for gel electrophoresis.
Line 79: Line 68:
|-
|-
|1
|1
-
|hPCD : mCh : SP1AB (BL01)
+
|Kozak
-
|18.834
+
|9.47
-
|-
+
-
|2
+
-
|fshPCD : mCh : SP1AB (BL05)
+
-
|14.471
+
-
|-
+
-
|3
+
-
|flyPCD : mCh : SP1A (BL09)
+
-
|7.302
+
-
|-
+
-
|4
+
-
|hPCD : BL01 (BL12)
+
-
|10.84
+
-
|-
+
|}
|}

Revision as of 16:35, 26 March 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:

  • Kozak
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


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 Kozak 9.47



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