User:Behzad Damadzadeh/Notebook/PcTF Subcloning in E-coli/2012/04/06: Difference between revisions
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'''Assemblies''' | |||
# BD112 (final assembly 1): BD111/(X/P)/1138 + BBa_I712074/(S/P)46 | |||
# BD113 (final assembly 2): BD111/(X/P)/1138 + BBa_I719005/(S/P)23 | |||
''' | [[Image:T7-RBS-PcTF-Assembly.jpg|thumb|350px|BD112 and BD113 assembly]] | ||
# | > Digests (Fermentas FD)<br> | ||
{| class="wikitable" width=400px | |||
| Plasmid DNA || 20.0 μl* | |||
|- | |||
| Fermentas FastDigest enzyme 1 || 1.0 μl | |||
|- | |||
| Fermentas FastDigest enzyme 2 || 1.0 μl | |||
|- | |||
| 10x FastDigest buffer + green loading dye || 3.0 μl | |||
|- | |||
| dH<sub>2</sub>O || 5.0 μl | |||
|- | |||
| || 30.0 μl total | |||
|- | |||
| colspan="2" | *For low yield DNA, use up to 25 μL; decrease dH<sub>2</sub>O accordingly.<br>Mix the reaction(s) thoroughly by flicking the tube.<br>Incubate at 37°C for 30 minutes. | |||
|} | |||
'''Separate the fragments via gel electrophoresis and purify the fragments''' | |||
[[Image:PcTF-RBS-Digestion-Gel-Behzad4-4-12.jpg|thumb|350px|White dashed lines border where the gel was cut to excise vector fragment (RBS) and insert fragment (PcTF).]] | |||
# Make a 0.8% gel: add 0.5 g agarose to ~60 ml 1x TAE buffer in a glass flask. | |||
# Mix by swirling and microwave for 40 seconds. Mix by swirling again (to eliminate air pockets and prevent boiling-over) and microwave for 40 seconds. | |||
# Set up a gel mold and comb. Make sure the teeth are the right size to hold 30 μL of sample. | |||
# Add 6 μl of 10 mg/ml ethidium bromide (etBr) to the agarose for a final concentration of ~0.8 μg/mL etBr. Mix by swirling (avoid making bubbles). | |||
# Pour the gel into the gel mold. Allow it to cool until it becomes opaque. | |||
# Fill a gel electrophoresis chamber with 1x TAE. | |||
# Remove the comb from the gel and carefully submerge the gel into the filled electrophoresis chamber. | |||
# Carefully pipette 15 μL pre-made 1 kb ladder mix into the first empty well and the DNA samples into the other empty wells. | |||
# Connect the electrical leads so that the positive end is at the bottom (DNA migrates to the positive end). Run the gel at 100 V. | |||
# Stop the gel when the yellow dye (Orange G) reaches the desired place on the gel (~1 hr.). | |||
# Remove the gel from the chamber and photograph under UV light. | |||
# Use a scalpel to cut the appropriate sized band(s) from the gel, place each gel slice in a 1.5 mL tube, and purify the DNA with Zymoclean Gel DNA Recovery Kit(Follow the protocol inside the kit box). | |||
# 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: | |||
PcTF: 15.512 (ng/μl), 2.123 (A260/A280) | |||
RBS: 3.201 (ng/μl), 2.462 (A260/A280) |
Revision as of 13:37, 7 April 2012
PcTF Subcloning in E. coli | <html><img src="/images/9/94/Report.png" border="0" /></html> Main project page <html><img src="/images/c/c3/Resultset_previous.png" border="0" /></html>Previous entry<html> </html>Next entry<html><img src="/images/5/5c/Resultset_next.png" border="0" /></html> | ||||||||||||||
06/04/12Assemblies
> Digests (Fermentas FD)
Separate the fragments via gel electrophoresis and purify the fragments
Biotek Take3 Result: PcTF: 15.512 (ng/μl), 2.123 (A260/A280) RBS: 3.201 (ng/μl), 2.462 (A260/A280) |