Admin: Created page with "= TFB Protocol for DH5a
= #Inoculate a single colony from an LB plate into 5ml of LB medium in a plating tube OR inoculate about 1 µL of competent ''E coli ''i..."

2021-02-01T19:42:07Z

Created page with "= TFB Protocol for DH5a = #Inoculate a single colony from an LB plate into 5ml of LB medium in a plating tube OR inoculate about 1 µL of competent ''E coli ''i..."

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= TFB Protocol for DH5a =

#Inoculate a single colony from an LB plate into 5ml of LB medium in a plating tube OR inoculate about 1 µL of competent ''E coli ''into 5 mL LB broth. Incubate overnight at 37°C with shaking (approximately 225rpm).'' ''
#Subculture the overnight culture 1:100 by inoculating 5 mL into 250 mL of LB supplemented with 20mM MgSO4 (0.6 g). Grow the cells in a 1 L flask until the OD600 reaches 0.4-0.6 (usually 5-6 hours, but the time may vary).
#Pellet the cells by centrifugation at 4,500 x g for 5 minutes at 4°C. For a 250 mL culture, use two 250ml centrifuge bottles in a large rotor. 
#Gently resuspend the cell pellet in 0.4 original volume of ice-cold TFB1. For a 250ml subculture, use 100ml of TFB1 (50 mL/bottle). Combine the resuspended cells in one bottle. For the remaining steps, keep the cells on ice and chill all pipettes, tubes and flasks. 
#Incubate the resuspended cells on ice for 15 minutes. 
#Pellet the cells by centrifugation at 4,500 x g for 5 minutes at 4°C. 
#Gently resuspend the cells in 1/25 original volume of ice-cold TFB2. For a 250ml subculture, use 10ml of TFB2. 
#Incubate the cells on ice for 15-60 minutes, then aliquot 100 µL/tube for storage at -80°C. Quick-freeze the tubes. JM109 competent cells prepared by this method are stable for 1 year. 

'''Note:''' Competent cells may be conveniently frozen in aliquot of 100 µL cells per tube. I typically transform 50 µL cells with 2-10 µL of a ligation reaction, and you should get between 1x108 to 1x109 cfu's/ug DNA. 

 

= Recipes =

===== LB broth (PSI broth) =====

For 1 liter:

*5g Bacto yeast extract
*20g Bacto Tryptone
*5g Sodium Chloride (NaCl)

pH 7.6 with potassium hydroxide 

===== TFB1 =====

For 200ml:

*0.588g potassium acetate
*2.42g rubidium chloride
*0.294g calcium chloride
*2.0g manganese chloride
*30ml glycerol

pH 5.8 with dilute acetic acid 

===== TFB2 =====

For 100ml:

*0.21g MOPS
*1.1g calcium chloride
*0.121g rubidium chloride
*15ml glycerol

pH 6.5 with dilute NaOH

= CaCl2 Protocol for BL21 & ZYCY10P3S2T =
Prepare two simple solutions:
=== Solution 1 ===
*0.1 M CaCl2 (1.11g in 100 mL)

=== Solution 2 ===
*0.1 M CaCl2 + 15% glycerol (1.11g in 85 mL ddH2O + 15 mL glycerol)
 
Solutions may be autoclaved, but filter-sterilization has been shown to yield better results

== Protocol ==
The night before:
*Grow a culture of bacteria in 1-5 mL SOB (or LB) overnight
 
'''Ensure all solutions are ice cold
''' 
*Add 1-2 mL of overnight culture to 100 mL of fresh LB medium and record starting OD600
*Grow for 2-4 hours at 37oC with shaking, until culture OD600 is 0.4-0.6
*Pellet the culture by centrifuging at >4000g for 5-10 minutes at 4oC
*Re-suspend the pellet in 20 mL Solution 1 and incubate for 30 minutes on ice with gentle shaking
*Pellet the culture by centrifuging at >4000g for 5-10 minutes at 4oC
*Re-suspend the pellet in 5 mL Solution 2 and snap-freeze 100 uL aliquots in LN2 before storing in -80oC freezer

Competent e coli - Revision history

Admin: Created page with "= TFB Protocol for DH5a = #Inoculate a single colony from an LB plate into 5ml of LB medium in a plating tube OR inoculate about 1 µL of competent ''E coli ''i..."

Admin: Created page with "= TFB Protocol for DH5a
= #Inoculate a single colony from an LB plate into 5ml of LB medium in a plating tube OR inoculate about 1 µL of competent ''E coli ''i..."