Team:UCL London/From the lab/Protocols

From 2009.igem.org

(Difference between revisions)
(New page: '''Competent Bacteria:''')
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'''Competent Bacteria:'''
'''Competent Bacteria:'''
 +
 +
:'''''Materials:'''''
 +
* 5×M9 salts in 500ml dH2O:
 +
** Na2HPO4--- 32g
 +
** KH2PO4 --- 7.5g
 +
** NaCl --- 1.25g
 +
** NH4Cl --- 2.5g
 +
 +
* Minimal media: ( In 50ml Falcon )
 +
** Melted bacteriological agar solution (< 50°C) --- 39ml
 +
** 5×M9 salt solution --- 10ml
 +
** 20% (w/v) D-glucose --- 1ml
 +
** 1M CaCl2 --- 5µl
 +
** 1M MgSO4 --- 100µl
 +
 +
* 0.1M CaCl2 / 15% glycerol: (In 59 ml Falcon)
 +
** 1M CaCl2 --- 5ml
 +
** 100% glycerol --- 7.5ml
 +
 +
: '''''Preparation:'''''
 +
:: Pour minimal media plates.
 +
:: 5x M9 salts.
 +
:: Prepare 100ml LB per strain.
 +
:: Prepare 50ml ice cold 0.1M CaCl2 / 15% glycerol per strain
 +
:: Pre-chill eppendorf tubes
 +
 +
: '''''Method:'''''
 +
# Streak cells on minimal agar plate. Incubate 37°C overnight.
 +
# Pick a colony into 5 ml LB + 100µl 1M MgSO4. Incubate 37°C overnight.
 +
# Inoculate 100ml LB in pre-warmed conical with 1ml of the 5ml O/N culture from Step 2.
 +
# Incubate 2 hrs in 37°C shaker until the cells at early log phase of growth curve (A600 ~ 0.3).
 +
# Transfer to chilled, sterile two 50ml Falcon tubes and incubate on ice for 10 min.
 +
# Cf 3300g 5 min in bechtop RmT. Cf.
 +
# Resus in 10ml ice cold 0.1M CaCl2 / 15% glycerol and incubate on ice 30 min.
 +
# Cf 3300g 5 min in benchtop RmT. Cf.
 +
# Resus in 1ml ice cold 0.1M CaCl2 / 15% glycerol. Transfer 100µl aliquots to pre-chilled, pre-labelled eppendorf tubes. Store -80°C.

Revision as of 08:43, 6 July 2009

Competent Bacteria:

Materials:
  • 5×M9 salts in 500ml dH2O:
    • Na2HPO4--- 32g
    • KH2PO4 --- 7.5g
    • NaCl --- 1.25g
    • NH4Cl --- 2.5g
  • Minimal media: ( In 50ml Falcon )
    • Melted bacteriological agar solution (< 50°C) --- 39ml
    • 5×M9 salt solution --- 10ml
    • 20% (w/v) D-glucose --- 1ml
    • 1M CaCl2 --- 5µl
    • 1M MgSO4 --- 100µl
  • 0.1M CaCl2 / 15% glycerol: (In 59 ml Falcon)
    • 1M CaCl2 --- 5ml
    • 100% glycerol --- 7.5ml
Preparation:
Pour minimal media plates.
5x M9 salts.
Prepare 100ml LB per strain.
Prepare 50ml ice cold 0.1M CaCl2 / 15% glycerol per strain
Pre-chill eppendorf tubes
Method:
  1. Streak cells on minimal agar plate. Incubate 37°C overnight.
  2. Pick a colony into 5 ml LB + 100µl 1M MgSO4. Incubate 37°C overnight.
  3. Inoculate 100ml LB in pre-warmed conical with 1ml of the 5ml O/N culture from Step 2.
  4. Incubate 2 hrs in 37°C shaker until the cells at early log phase of growth curve (A600 ~ 0.3).
  5. Transfer to chilled, sterile two 50ml Falcon tubes and incubate on ice for 10 min.
  6. Cf 3300g 5 min in bechtop RmT. Cf.
  7. Resus in 10ml ice cold 0.1M CaCl2 / 15% glycerol and incubate on ice 30 min.
  8. Cf 3300g 5 min in benchtop RmT. Cf.
  9. Resus in 1ml ice cold 0.1M CaCl2 / 15% glycerol. Transfer 100µl aliquots to pre-chilled, pre-labelled eppendorf tubes. Store -80°C.
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