User:Behzad Damadzadeh/Notebook/PcTF Subcloning in E-coli/2012/04/05: Difference between revisions
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'''Grow liquid cultures''' | '''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.<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). | # 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). | # 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). | ||
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# BD111 (Constructed gene)-Colony A , 69.251 (ng/μl) , 2.091 (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) | # 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.<br>''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)<br> | |||
{| class="wikitable" width=400px | |||
| Plasmid DNA || 2.0 μl* | |||
|- | |||
| EcoR1 || 1.0 μl | |||
|- | |||
| Pst1 || 1.0 μl | |||
|- | |||
| 10x FastDigest buffer + green loading dye || 1.5 μl | |||
|- | |||
| dH<sub>2</sub>O || 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. | |||
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Revision as of 20:05, 5 April 2012
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5/4/2012Culturing the colonies grew on agar plates on 4/4/2012
Grow liquid cultures
Extract the plasmid DNA: Qiagen Miniprep Kit' 1.5 hours Biotek Take3 Result:
Confirm the assembly Check the plates, grow cultures, and do minipreps 6 hours
> Digests (Fermentas FD)
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