User:Behzad Damadzadeh/Notebook/PcTF Subcloning in E-coli/2012/04/04: Difference between revisions
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# Remove the gel from the chamber and photograph under UV light. | # Remove the gel from the chamber and photograph under UV light. | ||
# Use a scalpel to cut the appropriate sized band(s) from the gel, place each gel slice in a 1.5 mL tube, and purify the DNA with Zymoclean Gel DNA Recovery Kit(Follow the protocol inside the kit box). | # Use a scalpel to cut the appropriate sized band(s) from the gel, place each gel slice in a 1.5 mL tube, and purify the DNA with Zymoclean Gel DNA Recovery Kit(Follow the protocol inside the kit box). | ||
# Measure the concentration of the purified fragment samples with a | # Measure the concentration of the purified fragment samples with a Biotek Take3 plate from the Stabenfeldt lab. Record the absorbance (A260), purity (A260/A280), and concentration (ng/μl) for each sample. | ||
Biotek Take3 Result: | |||
PcTF: 15.512 (ng/μl), 2.123 (A260/A280) | PcTF: 15.512 (ng/μl), 2.123 (A260/A280) | ||
RBS: 3.201 (ng/μl), 2.462 (A260/A280) | RBS: 3.201 (ng/μl), 2.462 (A260/A280) |
Revision as of 09:51, 5 April 2012
PcTF Subcloning in E. coli | <html><img src="/images/9/94/Report.png" border="0" /></html> Main project page Next entry<html><img src="/images/5/5c/Resultset_next.png" border="0" /></html> | ||||||||||||||||||||||||||||||||||||||
04/04/12General plan for assemblies
Assemblies
> Digests (Fermentas FD)
Separate the fragments via gel electrophoresis and purify the fragments
Biotek Take3 Result: PcTF: 15.512 (ng/μl), 2.123 (A260/A280) RBS: 3.201 (ng/μl), 2.462 (A260/A280)
Xng inser= (1123 / 2094) x 2 x 20 ng vector=22 Vectore= (20ng/3μL)= 7 μL Insert= (22ng/15μL)=2 μL
Transform bacteria with the ligated plasmids 30 minutes
Note: The negative control will show you the number of “background” colonies so that you can determine whether your transformation worked, or is just the result of vector self-ligation or selection failure.
DNA Re-Transformation of BBa_I712074, BBa_I719005, BBa_B0030
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