User:Behzad Damadzadeh/Notebook/PcTF Subcloning in E-coli/2012/11/16

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(11/16/12)
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# KAH150/pT7CFE1-CHis: KAH190/(E/S)/967 + "
# KAH150/pT7CFE1-CHis: KAH190/(E/S)/967 + "
# KAH151/pT7CFE1-CHis: KAH191/(E/S)/967 + "
# KAH151/pT7CFE1-CHis: KAH191/(E/S)/967 + "
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Note: KAH109 is being re-done as an internal control to trouble-shoot the ligation<br>
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Note: KAH111-KAH151 are all cut and purified already
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> Digest (Fermentas FD)<br>
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# pT7CFE1-CHis, E/P
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{| class="wikitable" border="0" cellspacing="3" <!-- Digest rxn. table -->
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|-valign="top"
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| <u>Reagent</u> || <u>Volume</u> || &nbsp;
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| rowspan="9" | [[Image:KAH_060311_gel1.tif|350px|Cloning digests 6/03/11]]<br>30 μL/lane, 1% agarose
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|-
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| DNA (plasmid) || 25.0 μL
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|-
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| 10x buffer || 3.0
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|-
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| EcoRI || 1.0
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|-
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| PstI || 1.0
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|-
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| dH<sub>2</sub>O || 0
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|-
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| &nbsp; || 30 μL --> 37°C/ ~30 min.
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|}

Revision as of 14:54, 16 November 2012

PcTF Subcloning in E. coli Main project page
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11/16/12

  • ---Karmella 13:51, 16 November 2012 (EST): Here is my suggested procedure for trying the PcTF fusion cloning...

Assemblies: Shown as Part/cuts/purified fragment size

  1. KAH109/pT7CFE1-CHis: KAH109/(E/P)/1132 + pT7CFE1-CHis/(E/P)/3600
  2. KAH111/pT7CFE1-CHis: KAH184/(E/S)/1123 + "
  3. KAH112/pT7CFE1-CHis: KAH187/(E/S)/1123 + "
  4. KAH115/pT7CFE1-CHis: KAH188/(E/S)/931 + "
  5. KAH148/pT7CFE1-CHis: KAH189/(E/S)/967 + "
  6. KAH150/pT7CFE1-CHis: KAH190/(E/S)/967 + "
  7. KAH151/pT7CFE1-CHis: KAH191/(E/S)/967 + "


Note: KAH109 is being re-done as an internal control to trouble-shoot the ligation
Note: KAH111-KAH151 are all cut and purified already


> Digest (Fermentas FD)

  1. pT7CFE1-CHis, E/P
Reagent Volume   Cloning digests 6/03/11
30 μL/lane, 1% agarose
DNA (plasmid) 25.0 μL
10x buffer 3.0
EcoRI 1.0
PstI 1.0
dH2O 0
  30 μL --> 37°C/ ~30 min.
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