Team:Newcastle/E. coli Competence

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{{Team:Newcastle/mainbanner}}
{{Team:Newcastle/mainbanner}}
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=Competent cells preparation=
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=Preparing competent ''E. coli''=
==Materials required==
==Materials required==
# Conical flask
# Conical flask
# 300ml of LB broth
# 300ml of LB broth
-
# 1/40 volume of an overnight culture of the desired strain
+
# 1/20 volume of an overnight culture of the desired strain
# Ice and ice bucket
# Ice and ice bucket
# eppendorf tubes
# eppendorf tubes
Line 14: Line 14:
# Ethanol dry ice
# Ethanol dry ice
# -80°C freezer
# -80°C freezer
 +
 +
===Preparation of 100 ml of TFB1===
 +
{|border=1
 +
|-
 +
!'''Number'''
 +
!'''Chemicals required'''
 +
!'''Volume'''
 +
|-
 +
|1
 +
|KAc
 +
|30 mM
 +
|-
 +
|2
 +
|CaCl2
 +
|10 mM
 +
|-
 +
|3
 +
|KCl
 +
|100 mM
 +
|-
 +
|4
 +
|Glycerol
 +
|15% (v/v)
 +
|-
 +
|5
 +
|Distill water
 +
|900 ml
 +
|-
 +
|6
 +
|500 mM MnCl2.4H2O
 +
|100 ml
 +
|}
 +
 +
===Preparation of 100 ml of TFB2===
 +
{|border=1
 +
|-
 +
!'''Number'''
 +
!'''Chemicals required'''
 +
!'''Volume'''
 +
|-
 +
|1
 +
|CaCl2
 +
|75 mM
 +
|-
 +
|2
 +
|KCl
 +
|10 mM
 +
|-
 +
|3
 +
|Glycerol
 +
|15% (v/v)
 +
|-
 +
|4
 +
|Distill water
 +
|900 ml
 +
|-
 +
|5
 +
|Na-MOPS pH 7.0
 +
|100 mM
 +
|}
==Procedures==
==Procedures==
-
# Inoculate 300 ml of LB broth in a conical flask and inoculate with 1/40 volume of an overnight culture of the desired strain.  
+
# Inoculate 300 ml of LB broth in a conical flask and inoculate with 1/20 volume of an overnight culture of the desired strain.  
-
# Grow the cell at 37°C in an incubator(with a shaking platform so as to mix the media equally amongst the cells).
+
# Grow the cell at 37°C, 200 rpm in an orbital incubator.
# Chill cells by placing the flask on ice and harvest by centrifugation at 4°C for 10 minutes.
# Chill cells by placing the flask on ice and harvest by centrifugation at 4°C for 10 minutes.
# Resuspend the pellet in 100 ml of ice cold TFB1 and gently shake the tubes whilst placed on ice.
# Resuspend the pellet in 100 ml of ice cold TFB1 and gently shake the tubes whilst placed on ice.
-
#Repeat the the above mentioned step and carefully resuspend pellet in 20 ml ice cold TFB2.
+
# Repeat the above mentioned step and carefully resuspend pellet in 20 ml ice cold TFB2.
-
# Aliquot 200 µl volumes of the cell suspension into cold sterile microfuge (1.5 ml) tubes and flash freeze in dry-ice  
+
# Aliquot 200 µl volumes of the cell suspension into cold sterile microfuge (1.5 ml) tubes and flash freeze in dry-ice .
-
# Store at -80°C
+
# Store at -80°C.
 +
 
 +
 
 +
'''Go back to our [[Team:Newcastle/Protocol list|Protocol List]]'''
{{Team:Newcastle/footer}}
{{Team:Newcastle/footer}}

Latest revision as of 13:50, 27 October 2010

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Contents

Preparing competent E. coli

Materials required

  1. Conical flask
  2. 300ml of LB broth
  3. 1/20 volume of an overnight culture of the desired strain
  4. Ice and ice bucket
  5. eppendorf tubes
  6. TFB1
  7. TFB2
  8. 1.5 ml Microfuge tubes
  9. Ethanol dry ice
  10. -80°C freezer

Preparation of 100 ml of TFB1

Number Chemicals required Volume
1 KAc 30 mM
2 CaCl2 10 mM
3 KCl 100 mM
4 Glycerol 15% (v/v)
5 Distill water 900 ml
6 500 mM MnCl2.4H2O 100 ml

Preparation of 100 ml of TFB2

Number Chemicals required Volume
1 CaCl2 75 mM
2 KCl 10 mM
3 Glycerol 15% (v/v)
4 Distill water 900 ml
5 Na-MOPS pH 7.0 100 mM

Procedures

  1. Inoculate 300 ml of LB broth in a conical flask and inoculate with 1/20 volume of an overnight culture of the desired strain.
  2. Grow the cell at 37°C, 200 rpm in an orbital incubator.
  3. Chill cells by placing the flask on ice and harvest by centrifugation at 4°C for 10 minutes.
  4. Resuspend the pellet in 100 ml of ice cold TFB1 and gently shake the tubes whilst placed on ice.
  5. Repeat the above mentioned step and carefully resuspend pellet in 20 ml ice cold TFB2.
  6. Aliquot 200 µl volumes of the cell suspension into cold sterile microfuge (1.5 ml) tubes and flash freeze in dry-ice .
  7. Store at -80°C.


Go back to our Protocol List

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