Transformation Protocol: Difference between revisions
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>YanWu (Created page with "#Ensure water bath at 42 C, warm SOC media in incubator at 37 C #Add 200 ng DNA into each tube of 100 uL cells (1-5 uL vector) #Incubate on ice for 30 minutes #Heat-shock for ...") |
>YanWu No edit summary |
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#Ensure water bath at 42 C, warm SOC media in incubator at 37 C | #Ensure water bath at 42 C, warm SOC media in incubator at 37 C | ||
#Add 200 ng DNA into each tube of 100 uL cells (1-5 uL vector) | #Add 200 ng DNA into each tube of 100 uL cells (1-5 uL vector). You want to use up all of your vector so as to get the best coverage. | ||
#Incubate on ice for 30 minutes | #Incubate on ice for 30 minutes | ||
#Heat-shock for 45 seconds @ 42 C | #Heat-shock for 45 seconds @ 42 C | ||
Line 6: | Line 6: | ||
#Rescue cells with 500 uL SOC per tube | #Rescue cells with 500 uL SOC per tube | ||
#Shake at 37 C for 1 hr in incubator | #Shake at 37 C for 1 hr in incubator | ||
#Spread up to 20 uL on at least 2 plates for each library | #Spread up to 20 uL on at least 2 plates for each library. For control spread 100 uL on each plate, using at least 2 plates. | ||
#Add all tubes into large culture flask (150 mL) grow overnight | #Add all tubes into large culture flask (150 mL) grow overnight | ||
#The next day, make at least 10 tubes of frozen stock from large culture and then maxi-prep |
Latest revision as of 23:17, 6 May 2015
- Ensure water bath at 42 C, warm SOC media in incubator at 37 C
- Add 200 ng DNA into each tube of 100 uL cells (1-5 uL vector). You want to use up all of your vector so as to get the best coverage.
- Incubate on ice for 30 minutes
- Heat-shock for 45 seconds @ 42 C
- Incubate on ice again for 2 minutes
- Rescue cells with 500 uL SOC per tube
- Shake at 37 C for 1 hr in incubator
- Spread up to 20 uL on at least 2 plates for each library. For control spread 100 uL on each plate, using at least 2 plates.
- Add all tubes into large culture flask (150 mL) grow overnight
- The next day, make at least 10 tubes of frozen stock from large culture and then maxi-prep