User:Behzad Damadzadeh/Notebook/PcTF Subcloning in E-coli/2012/04/05: Difference between revisions
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|style="background-color: #EEE"|[[Image:owwnotebook_icon.png|128px]]<span style="font-size:22px;">PcTF Subcloning in E. coli</span> | |style="background-color: #EEE"|[[Image:owwnotebook_icon.png|128px]]<span style="font-size:22px;">PcTF Subcloning in E. coli</span> | ||
|style="background-color: #F2F2F2" align="center"| | |style="background-color: #F2F2F2" align="center"|[[File:Report.png|frameless|link={{#sub:{{FULLPAGENAME}}|0|-11}}]][[{{#sub:{{FULLPAGENAME}}|0|-11}}|Main project page]]<br />{{#if:{{#lnpreventry:{{FULLPAGENAME}}}}|[[File:Resultset_previous.png|frameless|link={{#lnpreventry:{{FULLPAGENAME}}}}]][[{{#lnpreventry:{{FULLPAGENAME}}}}{{!}}Previous entry]] }}{{#if:{{#lnnextentry:{{FULLPAGENAME}}}}|[[{{#lnnextentry:{{FULLPAGENAME}}}}{{!}}Next entry]][[File:Resultset_next.png|frameless|link={{#lnnextentry:{{FULLPAGENAME}}}}]]}} | ||
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'''Culturing the colonies grew on agar plates on 4/4/2012''' | '''Culturing the colonies grew on agar plates on 4/4/2012''' | ||
# BBa_I712074 | # BBa_I712074 (Re-transformed gene) | ||
# BBa_I719005 | # BBa_I719005 (Re-transformed gene) | ||
# BBa_B0030 | # BBa_B0030 (Re-transformed gene) | ||
# BD111 | # BD111 (Constructed gene) | ||
'''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). | ||
# Grow the cultures for 7 hours in a shaking 37°C incubator.<br><br> | # 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: | |||
# 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.<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> | |||
[[Image:BD-111-Behzad-5-4-12.jpg|thumb|350px|White dashed lines border show the vector backbone and the BD-111 constructed gene(RBS + PcTF).]] | |||
{| 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 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' |
Latest revision as of 21:30, 26 September 2017
PcTF Subcloning in E. coli | Main project page Previous entry Next entry | |||||||||||||
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)
'The size of the constructed gene supposed to be: 1123 + 15 + 6 = 1144 bp' |