User:Behzad Damadzadeh/Notebook/PcTF Subcloning in E-coli/2012/11/20: Difference between revisions
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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... | ||
# | |||
---- | |||
'''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> || | |||
| 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 | |||
|- | |||
| || 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 --> | |||
| || 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 || --- || --- || --- || --- || --- || --- || --- || --- | |||
|- | |||
| || 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> </html>Next entry<html><img src="/images/5/5c/Resultset_next.png" border="0" /></html> | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
11/16/12
Assemblies: Shown as Part/cuts/purified fragment size
Measure concentration(s)
Ligations (trouble shooting suggestions)
|