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. For the recombinant plasmid (BD-111), compare the plates to estimate the ratio of “ligation” colonies to “negative control” colonies. (100 ligation colonies and 1 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. 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)

Confirm the assembly

Check the plates, grow cultures, and do minipreps 6 hours

  1. If the ratio is 10:1 or greater, great job! Pick 2 colonies for separate liquid cultures (see Day 1, Grow 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.
  2. Digest 2 uL of each DNA sample with EcoRI/ PstI and check via gel electrophoresis (1% agarose) to confirm the assembled construct size. You should see one fragment that is the backbone, and another fragment that equals the total size of the two BioBrick parts you assembled.

> Digests (Fermentas FD)

White dashed lines border show the vector backbone and the BD-111 constructed gene(RBS + PcTF).
Plasmid DNA 2.0 μl*
EcoR1 1.0 μl
Pst1 1.0 μl
10x FastDigest buffer + green loading dye 1.5 μl
dH2O 9.5 μl
  15.0 μl total
Incubate at 37°C for 30 minutes.
  1. Make the (1%) agarose glee and add 15μl of restricted DNA (PcTF & RBS) in one well and 10 μl of ladder.

'The size of the constructed gene supposed to be: 1123 + 15 + 6 = 1144 bp'

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