Maloof Lab:Ultra-competent E.coli: Difference between revisions
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<h3><font style="color:#F8B603;">Making ultra-competent E.coli for heat-shock transformation (Inoue method)</font></h3> | <h3><font style="color:#F8B603;">Making ultra-competent E.coli for heat-shock transformation (Inoue method)</font></h3> | ||
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Within this protocol: | |||
''' Making ultra-competent cells''' | |||
''' Making Inoue buffer''' | |||
''' Making SOB and SOC''' | |||
''' Transforming the cells''' | |||
''' Calculating transformation efficiency''' | |||
'''To make ultra-competent cells:''' | '''To make ultra-competent cells:''' | ||
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# Freeze the cells in liquid nitrogen | # Freeze the cells in liquid nitrogen | ||
# Store at -80°C | # Store at -80°C | ||
'''To make Inoue buffer:''' | '''To make Inoue buffer:''' | ||
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Ideally, "ultra-competent cells" will have a competency of about 10<sup>7</sup> or 10<sup>8</sup>. | Ideally, "ultra-competent cells" will have a competency of about 10<sup>7</sup> or 10<sup>8</sup>. | ||
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Revision as of 21:31, 26 August 2012
Room 2115 |
Making ultra-competent E.coli for heat-shock transformation (Inoue method)Within this protocol: Making ultra-competent cells Making Inoue buffer Making SOB and SOC Transforming the cells Calculating transformation efficiency
To make ultra-competent cells:
For 100mL Inoue buffer (more than enough for 100mL cells),
e.g. you count 1500 colonies on a plate
+ 1μL DNA @ 1μg/mL +600μL SOC 711μL → plated 10μL
1500 colonies / 0.014 = ~1x105 Ideally, "ultra-competent cells" will have a competency of about 107 or 108.
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