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

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==Protocol==
==Protocol==
'''''Preperation of Competent Cells'''''
'''''Day 1'''''
'''''Day 1'''''
*Inoculate 10ml of LB broth from a single colony of ''B.subtilis'' on an LB agar plate and grow overnight at 37<sup>o</sup>
*Inoculate 10ml of LB broth from a single colony of ''B.subtilis'' on an LB agar plate and grow overnight at 37<sup>o</sup>C
'''''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)
*Pre-warm 100ml of 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 OD<sub>600</sub> of 1.5.
*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.
*Once at an OD<sub>600</sub> of 1.5 the cells should be aliquoted into 4x50ml Falcon tubes and  centrifugation at 4000 g for 15 minutes at RT. After this, remove the supernatent and resuspend the pellets in 10ml of ddH<sub>2</sub>O. Combine the 4x resuspended pellets so we get 2x50ml falcon tubes and repeat spin. After this remove the supernatent and resuspend in 25ml of ddH<sub>2</sub> and repeat the spin.  
*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 the 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). To do this pipette 1ml of PEG into one pellet and resuspend, then pipette this suspension into the next pellet and resuspend.
*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.
*This suspension should then be seperated into 100ul samples and pipetted into 1.5ml eppendorff tubes. After pipetting each aliquote place into a tray of dry ice to rapidly freeze samples and store at -80<sup>o</sup>C.  
*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.
<br>
*Now add pulse the cells on the following settings:
'''''Electroporation'''''<br>
**Field Strength - 12kv/cm
*Remove a 100 μl aliquote and thaw on ice. While thawing cells add 500 ng of DNA into 10μl of water, gently mix and transfer to a pre-chilled 0.1 cm gap electroporation cuvette.
*Adjust the settings of the electroporator to the following:
**Field Strength - 12kv/cm (1.2kv)
**Capacitamce - 25µF
**Capacitamce - 25µF
**Resistance - 400 ohms  
**Resistance - 400 ohms  
**Pulse Length - 8msec
**Pulse Length - 8msec
*After transformation the cells should then be immediately diluted in 2ml of SOC broth and transferred to 15 ml Falcon centrifuge tubes.  
*After transformation dilute the cells in 2ml of SOC broth and transfer 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 should be centrifuged at 3000g for 15 minutes. After spinning the majority of the supernatent should be removed, leaving enough media to allow resuspension.
*This suspension should eb streaked out on an LB agar plate containing 100ug/ml of Spectinomycin.


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Revision as of 08:53, 13 August 2008

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Transformation Protocol 4

Reagents

-SOC broth, per litre: - 10ml

  • 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, per litre:-110ml

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

-10% PEG 6000-10ml

-One LB agar plate containing streptinomycin 100ug/ml

Protocol

Preperation of Competent Cells Day 1

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

Day 2

  • Pre-warm 100ml of 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.
  • Once at an OD600 of 1.5 the cells should be aliquoted into 4x50ml Falcon tubes and centrifugation at 4000 g for 15 minutes at RT. After this, remove the supernatent and resuspend the pellets in 10ml of ddH2O. Combine the 4x resuspended pellets so we get 2x50ml falcon tubes and repeat spin. After this remove the supernatent and resuspend in 25ml of ddH2 and repeat the spin.
  • After the last wash the 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). To do this pipette 1ml of PEG into one pellet and resuspend, then pipette this suspension into the next pellet and resuspend.
  • This suspension should then be seperated into 100ul samples and pipetted into 1.5ml eppendorff tubes. After pipetting each aliquote place into a tray of dry ice to rapidly freeze samples and store at -80oC.


Electroporation

  • Remove a 100 μl aliquote and thaw on ice. While thawing cells add 500 ng of DNA into 10μl of water, gently mix and transfer to a pre-chilled 0.1 cm gap electroporation cuvette.
  • Adjust the settings of the electroporator to the following:
    • Field Strength - 12kv/cm (1.2kv)
    • Capacitamce - 25µF
    • Resistance - 400 ohms
    • Pulse Length - 8msec
  • After transformation dilute the cells in 2ml of SOC broth and transfer 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 should be centrifuged at 3000g for 15 minutes. After spinning the majority of the supernatent should be removed, leaving enough media to allow resuspension.
  • This suspension should eb streaked out on an LB agar plate containing 100ug/ml of Spectinomycin.


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