NanoBio: Bacterial Transformation: Difference between revisions
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==Traditional Transformation== | |||
# Thaw competent cells (One Shot Chemically Competent TOP10 from Invitrogen, catalog # C4040-03) on ice. | # Thaw competent cells (One Shot Chemically Competent TOP10 from Invitrogen, catalog # C4040-03) on ice. | ||
# Place 10-15 µL cells in pre-chilled Eppendorf tubes. | # Place 10-15 µL cells in pre-chilled Eppendorf tubes. | ||
| Line 10: | Line 12: | ||
# Add all media to a selectable marker LB plate. Use glass beads to streak the plate. | # Add all media to a selectable marker LB plate. Use glass beads to streak the plate. | ||
# Incubate at 37 °C. Transformants should appear within 16 hrs. | # Incubate at 37 °C. Transformants should appear within 16 hrs. | ||
*'''[[User:CarolineAjo-Franklin|CAjoF]] 19:52, 29 January 2008 (CST)''': | |||
==Simultaneous Transformation== | |||
Note: this protocol is for a different line of competent cells than described above. | |||
# Thaw competent cells (Ultra BL21(DE#) Competent Cells from Edge BioSystems) on ice. | |||
# Place 10-15 µL cells in pre-chilled Eppendorf tubes. | |||
# Add 1.0 µL of each 1:10 diluted mini-prepped plasmid. | |||
#* Plan ahead so that each plasmid has different antibiotic resistance. | |||
#* If the concentrations of the mini-prepped plasmids are not similar, you may want to dilute so that the added plasmids are equinormal. | |||
#* You may also need to up concentrations of the miniprepped plasmids if they are particularly large (>10kb) | |||
#*The volume of plasmid should be 10-20% of the total volume. | |||
# Chill on ice for 10 min. | |||
#*Do not pipet or vortex. | |||
# Heat shock at 42 °C for 40 s. | |||
# Incubate on ice for 2 min. | |||
# Add 200 µL SOC medium (~10 x volume; Invitrogen), and shake at 37 °C for 1 hour. | |||
# Add all media to a LB plates with both appropriate antibiotics. Use glass beads to streak the plate. | |||
# Incubate at 37 °C. Transformants should appear within 16 hrs. | |||
#* It is ideal to also plate out each plasmid separately on appropriate selector LB plates to confirm that each plasmid may transform into this particular line of cells at the selected concentration. | |||
*'''[[User:CarolineAjo-Franklin|CAjoF]] 19:52, 29 January 2008 (CST)''': | *'''[[User:CarolineAjo-Franklin|CAjoF]] 19:52, 29 January 2008 (CST)''': | ||
Return to [[NanoBio:Protocols|Protocols]]. | Return to [[NanoBio:Protocols|Protocols]]. | ||
Revision as of 11:26, 11 June 2008
Traditional Transformation
- Thaw competent cells (One Shot Chemically Competent TOP10 from Invitrogen, catalog # C4040-03) on ice.
- Place 10-15 µL cells in pre-chilled Eppendorf tubes.
- Add 2 µL ligated vector or diluted mini-prepped plasmid (1:10 to 1:50 will work), and chill on ice for 30 min.
- The volume of plasmid should be 10-20% of the total volume.
- Do not pipet or vortex.
- Heat shock at 42 °C for 30 s.
- Incubate on ice for 2 min.
- Add 170 µL SOC medium (~10 x volume; Invitrogen), and shake at 37 °C for 15 min for Amp selection, or 1 hr for Chl, Kan, or Tet selection.
- If you are in a hurry, you can directly plate Amp resistant plasmids on Amp containing plates.
- Add all media to a selectable marker LB plate. Use glass beads to streak the plate.
- Incubate at 37 °C. Transformants should appear within 16 hrs.
- CAjoF 19:52, 29 January 2008 (CST):
Simultaneous Transformation
Note: this protocol is for a different line of competent cells than described above.
- Thaw competent cells (Ultra BL21(DE#) Competent Cells from Edge BioSystems) on ice.
- Place 10-15 µL cells in pre-chilled Eppendorf tubes.
- Add 1.0 µL of each 1:10 diluted mini-prepped plasmid.
- Plan ahead so that each plasmid has different antibiotic resistance.
- If the concentrations of the mini-prepped plasmids are not similar, you may want to dilute so that the added plasmids are equinormal.
- You may also need to up concentrations of the miniprepped plasmids if they are particularly large (>10kb)
- The volume of plasmid should be 10-20% of the total volume.
- Chill on ice for 10 min.
- Do not pipet or vortex.
- Heat shock at 42 °C for 40 s.
- Incubate on ice for 2 min.
- Add 200 µL SOC medium (~10 x volume; Invitrogen), and shake at 37 °C for 1 hour.
- Add all media to a LB plates with both appropriate antibiotics. Use glass beads to streak the plate.
- Incubate at 37 °C. Transformants should appear within 16 hrs.
- It is ideal to also plate out each plasmid separately on appropriate selector LB plates to confirm that each plasmid may transform into this particular line of cells at the selected concentration.
- CAjoF 19:52, 29 January 2008 (CST):
Return to Protocols.
