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

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The following list of parts are:
The following list of parts are:
-
 
-
 
* BL01  
* BL01  
* BL05  
* BL05  
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| rowspan="7" | [[Image:gel3_22_2013.jpg|200px|Stock digest 3/18/2013]] [[Image:gel3_22_2013_cutout.jpg|200px|Stock digest 3/18/2013]] <br>30 μL/lane, 1% agarose; [http://openwetware.org/wiki/Image:KAH_Fermentas_GeneRuler_1kbplus.jpg Ladder]
| rowspan="7" | [[Image:gel3_22_2013.jpg|200px|Stock digest 3/18/2013]] [[Image:gel3_22_2013_cutout.jpg|200px|Stock digest 3/18/2013]] <br>30 μL/lane, 1% agarose; [http://openwetware.org/wiki/Image:KAH_Fermentas_GeneRuler_1kbplus.jpg Ladder]
|-
|-
-
| DNA (plasmid) || 20.0 μL
+
| DNA (plasmid) || 15.0 μL
 +
|-
 +
| XbaI|| 1.0 μL
|-
|-
-
| 10x buffer || 3.0
+
| PstI|| 1.0 μL
|-
|-
-
| XbaI || 2.0
+
|10x FastDigest buffer + green loading dye  || 1.0 μL
|-
|-
-
| dH<sub>2</sub>O || 5.0
+
| dH<sub>2</sub>O || 10 μL
|-
|-
-
| &nbsp; || 30 μL --> 37°C/ ~15 min.
+
| &nbsp; || 30 μL --> 37°C/ ~10 min.
|}
|}

Revision as of 23:28, 22 March 2013

Engineering PC-TFs Main project page
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Summary

Cutting inserts to be placed in mammalian vector

  • Procedure

A) Get biobricks and enzymes from freezer.

SpeI, PstI, XbaI

The following list of parts are:

  • BL01
  • BL05
  • BL09
  • 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


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
10x FastDigest buffer + green loading dye 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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