User:Behzad Damadzadeh/Notebook/PcTF Subcloning in E-coli/2013/01/21: Difference between revisions
From OpenWetWare
(Autocreate 2013/01/21 Entry for User:Behzad_Damadzadeh/Notebook/PcTF_Subcloning_in_E-coli) |
(→Date) |
||
Line 7: | Line 7: | ||
<!-- ##### DO NOT edit above this line unless you know what you are doing. ##### --> | <!-- ##### DO NOT edit above this line unless you know what you are doing. ##### --> | ||
= | =01/21/2013= | ||
=Chemically Competent Cell Prep ''(BL21-DE3)''= | |||
Haynes Lab, 2013 | |||
' | Prepare these solutions ahead of time and chill at 4°C overnight:<br> | ||
# | 100 mM CaCl<sub>2</sub>, autoclaved<br> | ||
15% glycerol in sterile 100 mM CaCl<sub>2</sub><br><br> | |||
* First autoclave the 100mM CaCl<sub>2</sub><br><br> and then make 15% glycerol solution. (Do not autoclave the glycerol) | |||
# Grow untransformed cells on plain LB (streak for single colonies). | |||
# Inoculate 12.5 mL culture with colony from plate. Incubate with shaking at 37°C for 8 hours. | |||
# Prepare two 250 mL of plane LB Broth and autoclave. | |||
# Transfer 6.2 mL culture into 250 mL. Grow until absorbance of 0.6 A600 is reached. BL21 growth is slow, overnight incubation is recommended. (12hrs) | |||
# Aliquot 125 mL culture to 4 sterile large 250mL polycarbonate spin bottles. Chill on ice for 15 min. | |||
# Centrifuge cells at 4000 rpm for 10 min ate 4°C. (Dr. Wang's lab refrigerator centrifuge) | |||
# Resuspend cells in sterile, ice-cold 100 mM CaCl<sub>2</sub> by gentle pipetting (do not vortex!). Use 37.5 mL per 125mL culture (75 mL per 250 cell culture). Incubate on ice for 15 min. | |||
# Centrifuge cells at 4000 rpm for 10 min at 4°C. | |||
# Resuspend in sterile, ice-cold 100 mM CaCl<sub>2</sub>/15% glycerol (do not vortex!). Use 20 mL per 250 mL culture. | |||
*The cell pellets are very sticky. We need to pipette up and down for a long time to get them completely resuspended. | |||
# Leave on ice in a cold room/ fridge for 21 hours. | |||
# Freeze 200μL aliquots of cells in sterile microcentrifuge tubes. Store at -80°C. |
Revision as of 12:02, 22 January 2013
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> |
01/21/2013Chemically Competent Cell Prep (BL21-DE3)Haynes Lab, 2013 Prepare these solutions ahead of time and chill at 4°C overnight:
|