User:Behzad Damadzadeh/Notebook/PcTF Subcloning in E-coli/2012/04/05

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5/4/2012

Culturing the colonies grew on agar plates on 4/4/2012

  1. BBa_I712074 (Re-transformed gene)
  2. BBa_I719005 (Re-transformed gene)
  3. BBa_B0030 (Re-transformed gene)
  4. BD111 (Constructed gene)

Grow liquid cultures

  1. Label 15 ml sterile culture tube(s) appropriately. Fill each tube with 2 ml of LB growth medium + appropriate antibiotic (e.g., 100 μg/ml ampicillin).
  2. Using a sterile pipette tip, touch the bacterial streak (or pick up a single colony) and put the tip into the LB medium (bacterial end down).
  3. Grow the cultures for 7 hours in a shaking 37°C incubator.

Extract the plasmid DNA: Qiagen Miniprep Kit' 1.5 hours
To extract the plasmid DNA from the bacteria, perform a mini prep (refer to the Qiagen miniprep protocol). 2 ml of culture usually gives a yield of about 200 ng/μl (elution vol. = 75 μl). 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. 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:

  1. BBa_I712074 55.834 (ng/μl) , 2.083 (260/280)
  2. BBa_I719005 (Re-transformed gene) 55.422 (ng/μl) , 2.115 (260/280)
  3. BBa_B0030 (Re-transformed gene) 35.328 (ng/μl) , 2.114 (260/280)
  4. BD111 (Constructed gene)-Colony A , 69.251 (ng/μl) , 2.091 (260/280)
  5. BD111 (Constructed gene)-Colony B , 64.353 (ng/μl) , 2.085 (260/280)
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