IGEM:IMPERIAL/2008/Prototype/Wetlab/Transformation protocol 4: Difference between revisions

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'''''Day 2'''''
'''''Day 2'''''
*Collect 100ml of pre-warmed LB broth, remove 1ml and measure the OD<sub>600</sub> and keep as a blank. Then remove the rest of the overnight culture and add to the LB broth. Grow this culture at 37<sup>o</sup>C with vigorous aeration to an OD600 of 1.5-2.0. (Check every 1 hour)
*Collect 100ml of pre-warmed LB broth, remove 1ml and measure the OD<sub>600</sub> and keep as a blank. Then remove the rest of the overnight culture and add to the LB broth. Grow this culture at 37<sup>o</sup>C with vigorous aeration to an OD600 of 1.5-2.0. (Check every 1 hour)
*Once an OD<sub>600</sub> of 1.5-2.0 is reached the cells should be aliquoted into ....centrifugation tubes and  centrifugation at 3000 g for 10 minutes. After this the supernatent should be removed and washed in 100ml (okay volume?) de-ionised water and centrifuged again. Repeat this wash twice.
*Once an OD<sub>600</sub> of 1.5-2.0 is reached the cells should be aliquoted into 100ml centrifugation tubes and  centrifugation at 4000 g for 15 minutes. After this the supernatent should be removed and washed in 100ml of ddH<sub>2</sub>O and centrifuged again. Repeat this wash twice.
*After the last wash cells are resuspended in 30% polyethyleneglycol (PEG) 6000 to 1% of the original culture volume (So if our orginal volume was 100ml then we resuspend in 1ml).  
*After the last wash cells are resuspended in 30% polyethyleneglycol (PEG) 6000 to 1% of the original culture volume (So if our orginal volume was 100ml then we resuspend in 1ml).  
*The cells were then dispensed into 100 μl aliquots into 1.5ml eppendorff tubes and rapidly frozen on dry ice. Once frozen the cells are stored in the –80 freezer.  
*The cells are then dispensed into 100 μl aliquots into 1.5ml eppendorff tubes and rapidly frozen on dry ice. Once frozen the cells are stored in the –80 freezer.  
*Electroporation is performed using a Bio-Rad Gene Pulser and Pulse Controller. 100 μl Cells to be transformed were first thawed on ice, 500 ng DNA in 10μl of water added, gently mixed and transferred to a pre-chilled 0.2 cm gap electroporation cuvette.  
*100 μl of cells are then to be thawed on ice. While thawing cells add 500 ng of DNA into 10μl of water, gently mix and transfer to a pre-chilled 0.2 cm gap electroporation cuvette. Add cells to the electroporation cuvette.
*The cells were then immediately pulsed and diluted in 2ml SOC broth and transferred to 15 ml Falcon centrifuge tubes.  
*Now add pulse the cells on the following settings ....
*The cells should then be immediately diluted in 2ml of SOC broth and transferred to 15 ml Falcon centrifuge tubes.  
*Cells were incubated at 37<sup>o</sup>C with gentle shaking for 90 minutes to allow expression of the antibiotic resistance.
*Cells were incubated at 37<sup>o</sup>C with gentle shaking for 90 minutes to allow expression of the antibiotic resistance.
*After 90 minutes the cells were spuin down at 3000g for 10 minutes and the majority of the supernatent removed. Cells were then resuspended in the remaining media and streaked out onto an LB agar plate.  
*After 90 minutes the cells were spuin down at 3000g for 10 minutes and the majority of the supernatent removed. Cells were then resuspended in the remaining media and streaked out onto an LB agar plate.  


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Revision as of 08:51, 10 August 2008

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<html><a href=http://openwetware.org/wiki/IGEM:IMPERIAL/2008/Prototype><img width=50px src=http://openwetware.org/images/f/f2/Imperial_2008_Logo.png></img</a></html> Home The Project B.subtilis Chassis Wet Lab Dry Lab Notebook

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Transformation Protocol 4

Reagents

-SOC broth, per litre:

  • 2% tryptone (20g)
  • 0.5% yeast extract (5g)
  • 10mM NaCl (0.5844g)
  • 2.5mM KCL (0.186375g)
  • 10mM MgCl2 (2.033g)
  • 10mM MgSO4
  • 20mM glucose (3.6g)

-LB broth:

  • 1% tryptone,
  • 0.5% yeast extract
  • 0.5% NaCI

Protocol

Day 1

  • Inoculate 10ml of LB broth from a single colony of B.subtilis on an LB agar plate and grow overnight at 37o

Day 2

  • Collect 100ml of pre-warmed LB broth, remove 1ml and measure the OD600 and keep as a blank. Then remove the rest of the overnight culture and add to the LB broth. Grow this culture at 37oC with vigorous aeration to an OD600 of 1.5-2.0. (Check every 1 hour)
  • Once an OD600 of 1.5-2.0 is reached the cells should be aliquoted into 100ml centrifugation tubes and centrifugation at 4000 g for 15 minutes. After this the supernatent should be removed and washed in 100ml of ddH2O and centrifuged again. Repeat this wash twice.
  • After the last wash cells are resuspended in 30% polyethyleneglycol (PEG) 6000 to 1% of the original culture volume (So if our orginal volume was 100ml then we resuspend in 1ml).
  • The cells are then dispensed into 100 μl aliquots into 1.5ml eppendorff tubes and rapidly frozen on dry ice. Once frozen the cells are stored in the –80 freezer.
  • 100 μl of cells are then to be thawed on ice. While thawing cells add 500 ng of DNA into 10μl of water, gently mix and transfer to a pre-chilled 0.2 cm gap electroporation cuvette. Add cells to the electroporation cuvette.
  • Now add pulse the cells on the following settings ....
  • The cells should then be immediately diluted in 2ml of SOC broth and transferred to 15 ml Falcon centrifuge tubes.
  • Cells were incubated at 37oC with gentle shaking for 90 minutes to allow expression of the antibiotic resistance.
  • After 90 minutes the cells were spuin down at 3000g for 10 minutes and the majority of the supernatent removed. Cells were then resuspended in the remaining media and streaked out onto an LB agar plate.


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