Haynes Lab:Notebook/Engineering PC-TFs/2012/02/16: Difference between revisions
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* Meanwhile, get ice bucket and allow competent E. coli cells to thaw for five minutes. | * Meanwhile, get ice bucket and allow competent E. coli cells to thaw for five minutes. | ||
* Then follow transformation process: | * Then follow transformation process: | ||
> Warm five 100 μg/mL Amp agar plates at 37 °C | |||
> Thaw fresh tube of DH5α Turbo cells on ice | |||
> Resuspend invisible DNA pellet (stock DNA) in 10 μL dH2O | |||
> Add 0.5 μL stock DNA to 10 μL dH2O in 0.5 mL tubes | |||
> Add 30 μL of DH5α Turbo cells to DNA + dH2O | |||
--> Include #5 tube, water only, no DNA (negative control) | |||
> Incubate cells + DNA on ice for 5 min. | |||
> Label pre-warmed plates | |||
> Transfer cells + DNA onto agar | |||
> Add 10 - 15 sterile glass beads, shake, discard beads | |||
> Incubate plates at 37 °C overnight | |||
Revision as of 19:41, 17 February 2012
Engineering PC-TFs | <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> | ||||||||||||||||||||||||||||||||||||||||||||||||||||
Ligation/Assembly
Measure concentration of each fragment:
Results:
Assembly Scheme: Number beside vector and insert is number of base pairs
> Warm five 100 μg/mL Amp agar plates at 37 °C > Thaw fresh tube of DH5α Turbo cells on ice > Resuspend invisible DNA pellet (stock DNA) in 10 μL dH2O > Add 0.5 μL stock DNA to 10 μL dH2O in 0.5 mL tubes > Add 30 μL of DH5α Turbo cells to DNA + dH2O --> Include #5 tube, water only, no DNA (negative control) > Incubate cells + DNA on ice for 5 min. > Label pre-warmed plates > Transfer cells + DNA onto agar > Add 10 - 15 sterile glass beads, shake, discard beads > Incubate plates at 37 °C overnight
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