User:Behzad Damadzadeh/Notebook/PcTF Subcloning in E-coli/2012/11/20

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=Date=
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=11/16/12=
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'''List title'''
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*'''[[User:Karmella Haynes|---Karmella]] 13:51, 16 November 2012 (EST)''': Here is my suggested procedure for trying the PcTF fusion cloning...
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# List items
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 +
----
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'''Assemblies''': Shown as Part/cuts/purified fragment size
 +
# KAH109/pT7CFE1-CHis: KAH109/(E/P)/1132 + pT7CFE1-CHis/(E/P)/3600
 +
# KAH111/pT7CFE1-CHis: KAH184/(E/S)/1123 + "
 +
# KAH112/pT7CFE1-CHis: KAH187/(E/S)/1123 + "
 +
# KAH115/pT7CFE1-CHis: KAH188/(E/S)/931 + "
 +
# KAH148/pT7CFE1-CHis: KAH189/(E/S)/967 + "
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# KAH150/pT7CFE1-CHis: KAH190/(E/S)/967 + "
 +
# KAH151/pT7CFE1-CHis: KAH191/(E/S)/967 + "
 +
 
 +
* 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
 +
 
 +
 
 +
'''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:image.tif|350px|Cloning digest ]]<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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|}
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 +
 
 +
 
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'''Measure concentration(s)'''
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{| class="wikitable" border="0" cellspacing="3" <!-- [DNA] table -->
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|-
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| <u>Sample</u> || <u>OD260</u> || <u>260/280</u> || <u>ng/μL</u>
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|-
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| 1. pT7CFE1-CHis (E/P) || --- || --- || ---
 +
|}
 +
 
 +
 
 +
 
 +
'''Ligations''' (trouble shooting suggestions)
 +
* Note: Use 50 ng of vector this time (instead of 20)
 +
* Note: Use a 3:1 ratio of insert to vector... x μL insert = 3 * (bp of insert/ bp of vector) * 50 ng vector / (concentration of insert ng/μL)
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{| class="wikitable" border="0" cellspacing="3" <!-- Ligation rxn table -->
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| &nbsp;            || 1    || 2    || 3    || 4    || 5    || 6    || 7    || 8 
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|-
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| Insert DNA        || ---  || ---  || ---  || ---  || ---  || ---  || ---  || ---
 +
|-
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| Vector DNA        || ---  || ---  || ---  || ---  || ---  || ---  || ---  || ---
 +
|-
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| 2x lgn buf (Roche) || 5.0  || 5.0  || 5.0  || 5.0  || 5.0  || 5.0  || 5.0  || 5.0
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|-
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| T4 ligase (NEB)    || 1.0  || 1.0  || 1.0  || 1.0  || 1.0  || 1.0  || 1.0  || 1.0
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|-
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| dH<sub>2</sub>O    || ---  || ---  || ---  || ---  || ---  || ---  || ---  || ---
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|-
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| &nbsp;            || 10 μL || 10 μL || 10 μL || 10 μL || 10 μL || 10 μL || 10 μL || 10 μL
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|}
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 +
 
 +
* Add total ligation reaction to 30 μL DH5α-Turbo cells
 +
* Follow routine "fast" transformation procedure
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* Plate onto 100 ug/mL Amp agar plates

Revision as of 17:20, 20 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  
DNA (plasmid) 25.0 μL
10x buffer 3.0
EcoRI 1.0
PstI 1.0
dH2O 0
  30 μL --> 37°C/ ~30 min.


Measure concentration(s)

Sample OD260 260/280 ng/μL
1. pT7CFE1-CHis (E/P) --- --- ---


Ligations (trouble shooting suggestions)

  • Note: Use 50 ng of vector this time (instead of 20)
  • Note: Use a 3:1 ratio of insert to vector... x μL insert = 3 * (bp of insert/ bp of vector) * 50 ng vector / (concentration of insert ng/μL)
  1 2 3 4 5 6 7 8
Insert DNA --- --- --- --- --- --- --- ---
Vector DNA --- --- --- --- --- --- --- ---
2x lgn buf (Roche) 5.0 5.0 5.0 5.0 5.0 5.0 5.0 5.0
T4 ligase (NEB) 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0
dH2O --- --- --- --- --- --- --- ---
  10 μL 10 μL 10 μL 10 μL 10 μL 10 μL 10 μL 10 μL


  • Add total ligation reaction to 30 μL DH5α-Turbo cells
  • Follow routine "fast" transformation procedure
  • Plate onto 100 ug/mL Amp agar plates
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