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|style="background-color: #EEE"|[[Image:owwnotebook_icon.png|128px]]<span style="font-size:22px;"> Engineering PC-TFs</span>
|style="background-color: #FFCC00"|<br>[[Image:Hayneslab3.gif|200px|center]][[Image:Asu logo 3.gif|200px|center]]<span style="font-size:22px;"><br></span>
|style="background-color: #F2F2F2" align="center"|<html><img src="/images/9/94/Report.png" border="0" /></html> [[{{#sub:{{FULLPAGENAME}}|0|-11}}|Main project page]]<br />{{#if:{{#lnpreventry:{{FULLPAGENAME}}}}|<html><img src="/images/c/c3/Resultset_previous.png" border="0" /></html>[[{{#lnpreventry:{{FULLPAGENAME}}}}{{!}}Previous entry]]<html>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</html>}}{{#if:{{#lnnextentry:{{FULLPAGENAME}}}}|[[{{#lnnextentry:{{FULLPAGENAME}}}}{{!}}Next entry]]<html><img src="/images/5/5c/Resultset_next.png" border="0" /></html>}}
|style="background-color: #800000" align="center"|[[File:Report.png|frameless|link={{#sub:{{FULLPAGENAME}}|0|-11}}]][[{{#sub:{{FULLPAGENAME}}|0|-11}}|Main project page]]<br />{{#if:{{#lnpreventry:{{FULLPAGENAME}}}}|[[File:Resultset_previous.png|frameless|link={{#lnpreventry:{{FULLPAGENAME}}}}]][[{{#lnpreventry:{{FULLPAGENAME}}}}{{!}}Previous entry]]&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;}}{{#if:{{#lnnextentry:{{FULLPAGENAME}}}}|[[{{#lnnextentry:{{FULLPAGENAME}}}}{{!}}Next entry]][[File:Resultset_next.png|frameless|link={{#lnnextentry:{{FULLPAGENAME}}}}]]}}
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==Summary==
==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
 
{| {{table}}
|align="center" style="background:#f0f0f1;"|'''Stock Digest'''
<font size=3>''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 </font>
|}
 
''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.
 
{| {{table}} cellspacing="3" <!-- Digest rxn. table -->
|- valign="top"
| bgcolor=#cfcfcf | Reagent
| bgcolor=#cfcfcf | Volume
| 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) || 15.0 μL
|-
| XbaI|| 1.0 μL
|-
| PstI|| 1.0 μL
|-
| Green 10x FastDigest buffer || 1.0 μL
|-
| dH<sub>2</sub>O || 10 μL
|-
| &nbsp; || 30 μL --> 37°C/ ~10 min.
|}
 
 
{|border="1" cellpadding="5" cellspacing="0" align="left"
|-
! scope="col" style="background:#efefef;" | #
! scope="col" style="background:#efefef;" | Biobrick
! scope="col" style="background:#efefef;" | 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
|-
|}




Latest revision as of 22:34, 26 September 2017



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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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