User:Behzad Damadzadeh/Notebook/PcTF Subcloning in E-coli/2012/04/09: Difference between revisions
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''' | '''Grow liquid cultures''' | ||
# | #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) | ||
# If the ratio is 10:1 or greater, great job! Pick 2 colonies for separate liquid cultures. Grow for 5 - 6 hours.<br>''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.<br><br> | |||
''Extract the plasmid DNA: Qiagen Miniprep Kit''' ''1.5 hours''<br> | |||
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: | |||
# BD112 (Constructed gene)-Colony A , (ng/μl) , (260/280) | |||
# BD112 (Constructed gene)-Colony B , (ng/μl) , (260/280) | |||
# BD113 (Constructed gene)-Colony A , (ng/μl) , (260/280) | |||
# BD113 (Constructed gene)-Colony B , (ng/μl) , (260/280) |
Revision as of 18:04, 9 April 2012
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9/4/12Grow liquid cultures
Extract the plasmid DNA: Qiagen Miniprep Kit' 1.5 hours Biotek Take3 Result:
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