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4.Electroporation
=Transformation Protocol 4=


Reagents
==Reagents==
-SOC broth, per litre:
-'''''SOC broth, per litre:'''''<br>
2% tryptone (20g)
*2% tryptone (20g)
0.5% yeast extract (5g)  
*0.5% yeast extract (5g)  
10mM NaCl (0.5844g)
*10mM NaCl (0.5844g)
2.5mM KCL  (0.186375g)
*2.5mM KCL  (0.186375g)
10mM MgCl2  (2.033g)
*10mM MgCl2  (2.033g)
10mM MgSO4   
*10mM MgSO4   
20mM glucose (3.6g)   
*20mM glucose (3.6g)   


-LB broth  
-'''''LB broth:'''''
(1% tryptone, 0.5% yeast extract and 0.5% NaCI)
*1% tryptone,
1. B. subtilis strains were grown in LB broth at 37~ with vigorous aeration to an OD600 of 1.5-2.0.  
*0.5% yeast extract
2. Cells were harvested and washed three times in sterile cold de-ionised water by centrifugation at 3000 g for 10 minutes.
*0.5% NaCI
3. Cells resuspended in 30% polyethyleneglycol (PEG) 6000 to 1% of the original culture volume.  
 
4. The cells were then dispensed into 100 μl aliquots, rapidly frozen and stored at –70.  
==Protocol==
5. Where cells were harvested and suspended in either 15% glycerol or a range of PEG concentrations, they were used immediately.  
 
6. Electroporation was performed using a Bio-Rad Gene Pulser and Pulse Controller as described by Dower et aL (1988). 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.  
*B. subtilis strains were grown in LB broth at 37~ with vigorous aeration to an OD600 of 1.5-2.0.  
7. The cells were then immediately pulsed and diluted in 2ml SOC broth and transferred to 15 ml Falcon centrifuge tubes.  
*Cells were harvested and washed three times in sterile cold de-ionised water by centrifugation at 3000 g for 10 minutes.
8. Cells were incubated at 37oC with gentle shaking for 90 minutes to allow expression of the antibiotic resistance.  
*Cells resuspended in 30% polyethyleneglycol (PEG) 6000 to 1% of the original culture volume.  
*The cells were then dispensed into 100 μl aliquots, rapidly frozen and stored at –70.  
*Where cells were harvested and suspended in either 15% glycerol or a range of PEG concentrations, they were used immediately.  
*Electroporation was performed using a Bio-Rad Gene Pulser and Pulse Controller as described by Dower et aL (1988). 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.  
*The cells were then immediately pulsed and diluted in 2ml 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.  


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Revision as of 16:16, 6 August 2008

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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

  • B. subtilis strains were grown in LB broth at 37~ with vigorous aeration to an OD600 of 1.5-2.0.
  • Cells were harvested and washed three times in sterile cold de-ionised water by centrifugation at 3000 g for 10 minutes.
  • Cells resuspended in 30% polyethyleneglycol (PEG) 6000 to 1% of the original culture volume.
  • The cells were then dispensed into 100 μl aliquots, rapidly frozen and stored at –70.
  • Where cells were harvested and suspended in either 15% glycerol or a range of PEG concentrations, they were used immediately.
  • Electroporation was performed using a Bio-Rad Gene Pulser and Pulse Controller as described by Dower et aL (1988). 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.
  • The cells were then immediately pulsed and diluted in 2ml 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.


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