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

From OpenWetWare
Jump to navigationJump to search
(Autocreate 2012/11/20 Entry for User:Behzad_Damadzadeh/Notebook/PcTF_Subcloning_in_E-coli)
 
Line 7: Line 7:
<!-- ##### DO NOT edit above this line unless you know what you are doing. ##### -->
<!-- ##### DO NOT edit above this line unless you know what you are doing. ##### -->


=Date=
=11/16/12=


'''List title'''
*'''[[User:Karmella Haynes|---Karmella]] 13:51, 16 November 2012 (EST)''': Here is my suggested procedure for trying the PcTF fusion cloning...
# List items
 
----
'''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 + "
# 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>
* Note: KAH111-KAH151 are all cut and purified already
 
 
'''Digest (Fermentas FD)'''<br>
# pT7CFE1-CHis, E/P
 
{| class="wikitable" border="0" cellspacing="3" <!-- Digest rxn. table -->
|- valign="top"
| <u>Reagent</u> || <u>Volume</u> || &nbsp;
| rowspan="9" | <!--- [[Image:image.tif|350px|Cloning digest ]]<br>30 μL/lane, 1% agarose --->
|-
| DNA (plasmid) || 25.0 μL
|-
| 10x buffer || 3.0
|-
| EcoRI || 1.0
|-
| PstI || 1.0
|-
| dH<sub>2</sub>O || 0
|-
| &nbsp; || 30 μL --> 37°C/ ~30 min.
|}
 
 
 
'''Measure concentration(s)'''
{| class="wikitable" border="0" cellspacing="3" <!-- [DNA] table -->
|-
| <u>Sample</u> || <u>OD260</u> || <u>260/280</u> || <u>ng/μL</u>
|-
| 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)
 
{| class="wikitable" border="0" cellspacing="3" <!-- Ligation rxn table -->
| &nbsp;            || 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
|-
| dH<sub>2</sub>O    || ---  || ---  || ---  || ---  || ---  || ---  || ---  || ---
|-
| &nbsp;            || 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

Revision as of 14:20, 20 November 2012

PcTF Subcloning in E. coli <html><img src="/images/9/94/Report.png" border="0" /></html> Main project page
<html><img src="/images/c/c3/Resultset_previous.png" border="0" /></html>Previous entry<html>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</html>Next entry<html><img src="/images/5/5c/Resultset_next.png" border="0" /></html>

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
Retrieved from "https://openwetware.org/mediawiki/index.php?title=User:Behzad_Damadzadeh/Notebook/PcTF_Subcloning_in_E-coli/2012/11/20&oldid=657876"