User:Behzad Damadzadeh/Notebook/PcTF Subcloning in E-coli/2012/04/08: Difference between revisions
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= | =8/4/12= | ||
Continued from 6/4/12 | |||
''' | {| class="wikitable" width=400px align="right" | ||
# | | || Ligation || Negative Control | ||
|- | |||
| Insert DNA (X ng) || 2.0 μL || '''none''' | |||
|- | |||
| Vector DNA (20 ng) || 1.0 μL || 1.0 μL | |||
|- | |||
| 2x Roche Rapid Ligation buffer || 5 μl || 5 μL | |||
|- | |||
| New England Biolabs T4 ligase || 1.0 μl || 1.0 μL | |||
|- | |||
| dH<sub>2</sub>O || 1μl ||3 μL | |||
|- | |||
| || 10.0 μL total || 10 μL | |||
|- | |||
| colspan="3" | Mix the reaction(s) thoroughly by flicking the tube.<br>Incubate at room temperature for 10 minutes. | |||
|} | |||
'''Ligate (paste) the DNA fragments together''' ''15 minutes'' | |||
# Calculate how many ng of insert you need to get a 2:1 ratio of insert molecules to 50 ng vector molecules<br>''X ng insert = (bp insert / bp vector) x 2 x 50 ng vector'' | |||
# Calculate how many μL of insert and vector you will need for each ligation:<br>''X μL insert = desired ng insert ÷ insert concentration ng/μL'' (do the same for vector) | |||
# Set up your ligation reaction(s) in sterile 0.5 mL tubes as shown below: | |||
# Note: Because the low concentration of vector we planned to taked 20 ng vectore molecules instead of 50. | |||
BD-112 assembly: | |||
''Xng inser= (1144 / 3472) x 2 x 20 ng vector=14'' | |||
''Vectore= (20 ng/16μL)= 1.25 μL'' | |||
''Insert= (14ng/8μL)=2 μL'' | |||
BD-113 assembly: | |||
''Xng inser= (1144 / 2102) x 2 x 20 ng vector=21'' | |||
''Vectore= (20 ng/17μL)= 1.17 μL'' | |||
''Insert= (21ng/8μL)=2 μL'' | |||
<br><br> | |||
'''Transform bacteria with the ligated plasmids''' ''30 minutes'' | |||
# Warm selection agar plates at 37°C. | |||
# Incubate DH5α Turbo competent cells on ice just until thawed. Use 30 μL per ligation. | |||
# Add 30 μL thawed cells to the ligation reaction. Immediately place on ice and incubate for 10 min. (Do not heat shock; No 30 min. recovery is required for Amp resistance) | |||
# Label the pre-warmed plates with the antibiotic name, strain name, ligation (e.g., "BB part A insert + BB part B vector"), your initials, and the date. | |||
# Pipette the total volume of cells + ligation onto the agar; spread using sterile glass beads. | |||
# Incubate overnight at 37°C to get colonies | |||
Note: The negative control will show you the number of “background” colonies so that you can determine whether your transformation worked, or is just the result of vector self-ligation or selection failure. | |||
</div> |
Revision as of 16:50, 8 April 2012
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8/4/12Continued from 6/4/12
BD-112 assembly: Xng inser= (1144 / 3472) x 2 x 20 ng vector=14 Vectore= (20 ng/16μL)= 1.25 μL Insert= (14ng/8μL)=2 μL BD-113 assembly: Xng inser= (1144 / 2102) x 2 x 20 ng vector=21 Vectore= (20 ng/17μL)= 1.17 μL Insert= (21ng/8μL)=2 μL
Transform bacteria with the ligated plasmids 30 minutes
Note: The negative control will show you the number of “background” colonies so that you can determine whether your transformation worked, or is just the result of vector self-ligation or selection failure. |