Torsten Waldminghaus at 14:24, 9 December 2008

2008-12-09T14:24:46Z

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Streptomyces at 13:01, 14 August 2007

2007-08-14T13:01:21Z

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Streptomyces at 10:05, 10 August 2007

2007-08-10T10:05:37Z

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Streptomyces: New page: ==Protocols - Transformation by Electrophoration== {{Streptomyces}}
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2007-08-08T15:12:58Z

New page: ==Protocols - Transformation by Electrophoration==  {{Streptomyces}} {|cellspacing="5" cellpadding="10" style="background:#000; width: 800px;" |-valig...

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==Protocols - Transformation by Electrophoration==

{{Streptomyces}}
 
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==Transformation of Competent Cells by Electrophoration==

'''Description'''
 
Transform ''E.coli'' cells with plasmid/cosmid DNA using the method of electrophoration (inserting plasmids into ''E.coli'').

 
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| style="width: 150px;"| Approx. Duration: || style="width: 225px;"| || style="width: 75px;"| ||
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| || Preparation || ~30 minutes
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| || Making the competent cells || ~25 minutes
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| || Transformation by electrophoration || ~5 minutes
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| || Incubation step || ~1 hour
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| || Plating the transformed cells || ~10 minutes
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| || Whole protocol || ~2 hours 15 minutes
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'''Uses'''
 
Produce large quantities of plasmid DNA by transforming a fast growing ''E.coli'' strain - DH5α; used in conjunction with a mini prep. Change the plasmid DNA methylation state for compatibility with host bacteria during conjugations.

'''Prerequisites'''
 
You should know what the cells will be used for post-transformation in order to select the best ''E.coli'' strain to transform. You need to know what antibiotic resistance markers both the selected ''E.coli'' strain and the plasmid have. Also the approximate concentration of the DNA so the quantity used can be assessed.

'''Safety'''
 
General laboratory & molecular microbiology safety rules apply.

'''Requirements'''
 
LB/LB-NaCl, LBA/LBA-NaCl, 10% glycerol (sterile), 15mL falcon tube(s), electrophoration cuvette (for each transformation), micro-centrifuge tube(s) (MCT), incubating shaker @ 30/37°C, Electrophorator, falcon centrifuge, micro-centrifuge.
Optionally: Antibiotic(s)

'''Preparation'''
 
Pour 3-6 plates per transformation of LBA/LBA-NaCl + relevant antibiotics, and dry for ~1.5-2 hours. Place the 10% glycerol and DNA on ice. Prepare the electrophoration cuvette by ensuring it is clean, dry and ice cold. Set an MCT shaking incubator to 30/37°C. Label all tubes and plates to be used and have LB ready for the transformation.

'''Method'''
 
All parts are to be performed under sterile conditions, i.e. a Bunsen burner & kept chilled, except when using the electrophorator. This method is standard for DH5α transformations with pure (mini prepped) plasmid. For variations, please see the Notes.

Making the cells competent:
# After sub-culturing decant the 10mL culture into a 15mL falcon tube.
# Using the appropriate centrifuge in the cold room, spin the cells at 5000g for 3 minutes.
# Decant the supernatant and re-suspend the pellet in 1mL ice cold 10% glycerol, then top-up to 10mL.
# Repeat steps 2-3-2 in this order then progress to step 5.
# Re-suspend the pellet in 10% glycerol to a final volume of ~50µL.
# Place the cell suspension on ice.

Transformation by electrophoration:
# In an ice cold cuvette pipette ~50µL of the competent cells.
# Quickly add ~3µL of the plasmid DNA to the competent cells in the electrophoration cuvette.
# Without delay pulse the cells using the electrophorator.
# Very quickly add 1mL LB to the cuvette.
# Transfer the cells by pipette to a clean MCT and incubate for 1 hour @ 37°C.

Plating the transformed cells:
# Pipette various amounts of the cells onto each LBA+antibiotics plate, 50 - 150µL and spread with a flamed glass spreader.
# Dry the plates so the liquid is not visible.
# Incubate @ 37°C overnight.

'''Notes'''
 
Set the BioRad MicroPulser to the correct setting for the cuvettes being used, i.e. EC1 for BioRad brown capped (1mm) cuvettes or EC2 for green capped (2mm) cuvettes.
The whole procedure is best done as quickly as possible to avoid lysis of the cells and therefore degradation of the plasmid DNA when it is added.
The crucial steps in the success of a transformation by electrophoration are: 1) Thorough washing of the cells to remove all salts; 2) Re-suspension of the pellet and therefore correct cell concentration, 10mL culture in a final volume of ~50µL; 3) Limit the time between shocking the cells and adding the LB/LB-NaCl.
Ideally the time measurement of the pulse should be around 5.0ms (check by pressing measurements>time on the MicroPulser), this indicates the correct concentration of living cells with small amounts of electrolytes.
The transformation of pure (mini prepped) plasmids tends to be more efficient than transforming with a ligation mix. This affects two points: 1) how much DNA to transform with; 2) what volume of cells to plate. Generally the rule is to slightly increase the volume of DNA for ligations: ~3-6µL; and the amount plated: 50-300µL per plate.
Depending on the strain of ''E.coli'' being used, the incubator needs to be set accordingly, i.e. DH5α @ 37°C and BW25113 @ 30°C.
Another consideration with the strain is its growth rate; ET12567 doubling rate is slightly longer than DH5α. Thus the subculture needs a longer incubation (up to 4 hours) and the plates a few extra hours.


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Streptomyces:Protocols/Transformation by Electroporation - Revision history

Torsten Waldminghaus at 14:24, 9 December 2008

Streptomyces at 13:01, 14 August 2007

Streptomyces at 10:05, 10 August 2007

Streptomyces: New page: ==Protocols - Transformation by Electrophoration== {{Streptomyces}} {|cellspacing="5" cellpadding="10" style="background:#000; width: 800px;" |-valig...

Streptomyces: New page: ==Protocols - Transformation by Electrophoration== {{Streptomyces}}
{|cellspacing="5" cellpadding="10" style="background:#000; width: 800px;" |-valig...