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

PcTF Subcloning in E. coli

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5/4/2012 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. Biotek Take3 Result: BBa_I712074 55.834 (ng/μl) , 2.083 (260/280) BBa_I719005 (Re-transformed gene) 55.422 (ng/μl) , 2.115 (260/280) BBa_B0030 (Re-transformed gene) 35.328 (ng/μl) , 2.114 (260/280) 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 minipreps 6 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 10 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'