User:Behzad Damadzadeh/Notebook/PcTF Subcloning in E-coli/2012/11/16: Difference between revisions
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# KAH151/pT7CFE1-CHis: KAH191/(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 | # pT7CFE1-CHis, E/P | ||
{| class="wikitable" border="0" cellspacing="3" <!-- Digest rxn. table --> | {| class="wikitable" border="0" cellspacing="3" <!-- Digest rxn. table --> | ||
|-valign="top" | |- valign="top" | ||
| <u>Reagent</u> || <u>Volume</u> || | | <u>Reagent</u> || <u>Volume</u> || | ||
| rowspan="9" | <!--- [[Image:image.tif|350px|Cloning digest ]]<br>30 μL/lane, 1% agarose ---> | | rowspan="9" | <!--- [[Image:image.tif|350px|Cloning digest ]]<br>30 μL/lane, 1% agarose ---> | ||
Line 46: | Line 45: | ||
| || 30 μL --> 37°C/ ~30 min. | | || 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 12:22, 16 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)
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