Haynes Lab:Notebook/Engineering PC-TFs/2014/03/20: Difference between revisions
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==Summary== | ==Summary== | ||
* | *Ligation with CMV and MV9 using DH5α-T and BL21 cells | ||
{| class="wikitable" width=400px align="right" | |||
| || Ligation || Negative Control | |||
|- | |||
| Insert DNA (X ng) || 4μL || '''none''' | |||
|- | |||
| Vector DNA (50 ng) || 1μL || same | |||
|- | |||
| 2x Roche Rapid Ligation buffer || 6.0 μl || same | |||
|- | |||
| New England Biolabs T4 ligase || 1.0 μl || same | |||
|- | |||
| dH<sub>2</sub>O || 0μL || 0μL + ''Insert'' μL | |||
|- | |||
| || 12.0 μL total || same | |||
|- | |||
| colspan="3" | Mix the reaction(s) thoroughly by flicking the tube.<br>Incubate at room temperature for 10 minutes. | |||
|} | |||
*Repeat once again for BL21. 6μL of buffer was used because the total volume with no water exceeded 10μL. | |||
# 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 CMV = (bp CMV = 588 / bp MV9 = 5291) x 2 x 50 ng MV9 = 11.1132ng insert (CMV)'' | |||
# Calculate how many μL of insert and vector you will need for each ligation:<br>''X μL CMV = 11.1132ng CMV ÷ CMV concentration = 2.767ng/μL = 4.016μL'' <br>''X μL vector = 50 ng MV9 ÷ MV9 concentration = 49.159ng/μL = 1.0171μL'' | |||
# Set up your ligation reaction(s) in sterile 0.5mL tubes as shown here: | |||
<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 50 μL per ligation. | |||
# Add 50 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> | |||
<br><br> | |||
''''Transforming bacteria with the ligated plasmids in BL21'''' | |||
# Warm selection agar plates at 37°C. | |||
# Incubate BL21 competent cells on ice just until thawed. Use 50 μL per ligation. | |||
# Add 50 μL thawed cells to the ligation reaction. Immediately place on ice and incubate for 10 min. | |||
# Heat shock at 45°C for 45 seconds. | |||
# Immediately place on ice for 5 minutes. | |||
# Add 750μL SOC medium to two new 1.5mL autoclaved tubes. Label. | |||
# Transfer all cells and DNA to respectively labeled SOC medium tube. Place back on ice. | |||
# Tape both tubes horizontally to the shaking part (great science nomenclature) of the incubator for 30 minute recovery. | |||
# Pipette volume onto labeled and warmed agar plates. Spread using sterile glass beads. | |||
# Incubate overnight at 37°C to get colonies | |||
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Revision as of 15:50, 20 March 2014
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Summary
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.
'Transforming bacteria with the ligated plasmids in BL21'
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