User:Behzad Damadzadeh/Notebook/PcTF Subcloning in E-coli/2012/04/09

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PcTF Subcloning in E. coli Main project page
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9/4/12

Grow liquid cultures

  1. For the recombinant plasmids BD-112 and BD113, compare the plates to estimate the ratio of “ligation” colonies to “negative control” colonies. (more than 100 ligation colonies on BD-112 and many colonies on BD-113 plate and no negative control colony were observed)
  2. If the ratio is 10:1 or greater, great job! Pick 2 colonies for separate liquid cultures. Grow for 5 - 6 hours.
    If the ratio is less than 10:1, pick more colonies or trouble shoot and repeat the ligation & transformation.
  3. 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).
  4. 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).
  5. 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. BD112 (Constructed gene)-Colony A , 239.792 (ng/μl) , 2.198 (260/280)
  2. BD112 (Constructed gene)-Colony B , 251 (ng/μl) , 2.396 (260/280)
  3. BD113 (Constructed gene)-Colony A , 148.635 (ng/μl) , 2.8 (260/280)
  4. BD113 (Constructed gene)-Colony B , 120.331 (ng/μl) , 3.558 (260/280)
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