Culturing the colonies grew on agar plates on 4/4/2012
BBa_I712074 (Re-transformed gene)
BBa_I719005 (Re-transformed gene)
BBa_B0030 (Re-transformed gene)
BD111 (Constructed gene)
Grow liquid cultures
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)
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.
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).
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).
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.
BD111 (Constructed gene)-Colony A , 69.251 (ng/μl) , 2.091 (260/280)
BD111 (Constructed gene)-Colony B , 64.353 (ng/μl) , 2.085 (260/280)
Confirm the assembly
Check the plates, grow cultures, and do minipreps6 hours
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.
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.
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'
PcTF Subcloning in E. coli
