Team:Groningen/CaCl2 Competent Cells

From 2010.igem.org

(Difference between revisions)
(New page: '''Day 1:''' -Inoculate 5 ml LB( TY) medium with ''E.coli'' from glycerol stock -Grow overnight at 37 oC '''Day 2:''' -Inoculate 20 ml LB medium with 200 μl overnight culture ...)
 
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-Inoculate 5 ml [[LB( TY) medium]] with ''E.coli'' from glycerol stock  
-Inoculate 5 ml [[LB( TY) medium]] with ''E.coli'' from glycerol stock  
-
-Grow overnight at 37 oC
+
-Grow overnight at 37 ºC
'''Day 2:'''  
'''Day 2:'''  
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-Inoculate 20 ml LB medium with 200 μl overnight culture  
-Inoculate 20 ml LB medium with 200 μl overnight culture  
-
-Grow at 37 oC until OD600 = 0,3-0,5(+/- 2 hours)  
+
-Grow at 37 ºC until OD600 = 0,3-0,5(+/- 2 hours)  
-
-Spin down 5 minute at 4000 rpm at 4 oC
+
-Spin down 5 minute at 4000 rpm at 4 ºC
-Resuspend in 10 ml chilled 0,1 M CaCl2 (from here, keep on ice! )  
-Resuspend in 10 ml chilled 0,1 M CaCl2 (from here, keep on ice! )  
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-Divide 100 μl aliquots  
-Divide 100 μl aliquots  
-
-Store competent cells in - 80 oC
+
-Store competent cells in - 80 ºC
'''Transformation protocol'''
'''Transformation protocol'''
-
Melt agar in autoclave or microwave. Cool down to 60 oC. One jar ( 100mL ) is enough for 7 plates.
+
Melt agar in autoclave or microwave. Cool down to 60 ºC. One jar ( 100mL ) is enough for 7 plates.
-Add 10 μl of ligation product to 100 μl of cells ( don't for get controls! )
-Add 10 μl of ligation product to 100 μl of cells ( don't for get controls! )
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-Incubate 30 min on ice (make agar plates)
-Incubate 30 min on ice (make agar plates)
-
-Incubate for 1 min at 42 oC
+
-Incubate for 1 min at 42 ºC
-Incubate cells on ice for 2 min
-Incubate cells on ice for 2 min
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-Add 400 μl of [[SOC medium]]
-Add 400 μl of [[SOC medium]]
-
-Incubate for 30 min at 37 oC on shaker
+
-Incubate for 30 min at 37 ºC on shaker
-Spread 100-300 μl onto a plate made with appropriate antibiotic
-Spread 100-300 μl onto a plate made with appropriate antibiotic
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*If you expect the trasformation to be inefficient, concentrate cells by spinning the 500 μl and resuspending the pellet in 250 μl, which you will use for plating.
*If you expect the trasformation to be inefficient, concentrate cells by spinning the 500 μl and resuspending the pellet in 250 μl, which you will use for plating.
-
[[Growing streptomyces]]
+
'''Glycerol stock'''
 +
 
 +
Materials
 +
 
 +
*40% glycerol solution
 +
*Cryogenic vials
 +
 
 +
Methods
 +
 
 +
*Add 1 ml of 40% glycerol in H2O to a cryogenic vial
 +
*Add 1 ml sample from the culture of the bacteria to be stored( or,take 1 ml culture and add 300 μl of the 87% glycerol)
 +
*Gently vortex the cryogenic vial to ensure the culture and glycerol is well-mixed
 +
*Alternatively, pipet to mix
 +
*Use a tough spot to put the name of the strain or some useful identifier on the top of the vial
 +
*On the side of the vial list all relavant information-part,vector,strain, date,researcher,etc
 +
*Store in a freezer box in a -80 ºC freezer.Remember to record where vial is stored for fast retrieval later
 +
 
 +
Note: Ideally, your culture should be in logarithmic growth phase

Latest revision as of 22:44, 24 August 2010

Day 1:

-Inoculate 5 ml LB( TY) medium with E.coli from glycerol stock

-Grow overnight at 37 ºC

Day 2:

-Inoculate 20 ml LB medium with 200 μl overnight culture

-Grow at 37 ºC until OD600 = 0,3-0,5(+/- 2 hours)

-Spin down 5 minute at 4000 rpm at 4 ºC

-Resuspend in 10 ml chilled 0,1 M CaCl2 (from here, keep on ice! )

-Inoculate on ice for 20 minutes

-Spin down as before

-Remove supernatant

-Resuspend in 2 ml 0,1 M CaCl2 /10% glycerol

-Divide 100 μl aliquots

-Store competent cells in - 80 ºC

Transformation protocol

Melt agar in autoclave or microwave. Cool down to 60 ºC. One jar ( 100mL ) is enough for 7 plates.

-Add 10 μl of ligation product to 100 μl of cells ( don't for get controls! )

-Incubate 30 min on ice (make agar plates)

-Incubate for 1 min at 42 ºC

-Incubate cells on ice for 2 min

-Add 400 μl of SOC medium

-Incubate for 30 min at 37 ºC on shaker

-Spread 100-300 μl onto a plate made with appropriate antibiotic

-Grow overnight at 37 °C.

-Save the rest of the transformants in liquid culture at 4 °C. If nothing appears on your plate, you can spin this down, resuspend in enough medium to spread on one plate and plate it all. This way you will find even small numbers of transformants.

  • If you expect the transformation to be very efficient (for instance using undigested plasmid DNA) first make dilution (1:10 or 1:100 ) and plate 250 μl of this
  • If you expect the trasformation to be inefficient, concentrate cells by spinning the 500 μl and resuspending the pellet in 250 μl, which you will use for plating.

Glycerol stock

Materials

  • 40% glycerol solution
  • Cryogenic vials

Methods

  • Add 1 ml of 40% glycerol in H2O to a cryogenic vial
  • Add 1 ml sample from the culture of the bacteria to be stored( or,take 1 ml culture and add 300 μl of the 87% glycerol)
  • Gently vortex the cryogenic vial to ensure the culture and glycerol is well-mixed
  • Alternatively, pipet to mix
  • Use a tough spot to put the name of the strain or some useful identifier on the top of the vial
  • On the side of the vial list all relavant information-part,vector,strain, date,researcher,etc
  • Store in a freezer box in a -80 ºC freezer.Remember to record where vial is stored for fast retrieval later

Note: Ideally, your culture should be in logarithmic growth phase