Preparing chemically competent cells: Difference between revisions

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==Materials==
*Plate of cells to be made competent
*[[TSS]] buffer
*LB media
*Ice
==Glassware & Equipment
*Falcon tubes
*500μl Eppendorf tubes, on ice
*5ml pipete
*200ml conical flask
*Culture tube
*Repeating pipettor, tip that can make 100μl aliquots.
==Preparation==
==Preparation==
#Grow a 5 - 25 mL culture to an OD<sub>600</sub> of 0.2 - 0.5. (You will get a very small pellet if you grow 25ml to OD<sub>600</sub> 0.2)
#Grow a 5ml overnight culture of cells in LB media using a culture tube.  In the morning, dilute this culture back into 25-50ml of fresh LB media in a 200ml conical flask.  You should aim to dilute the overnight culture by at least 1/500.
#Grow the culture to an OD<sub>600</sub> of 0.2 - 0.5. (You will get a very small pellet if you grow 25ml to OD<sub>600</sub> 0.2)
#Put eppendorf tubes on ice now so that they are cold when cells are aliquoted into them later.  If your culture is Xml, you will need X tubes.  At this point you should also make sure that your [[TSS]] is being chilled (it should be stored at 4C but if you have just made it fresh then put it in an ice bath).
#Split the culture into two 50ml falcon tubes.
'''All subsequent steps should be carried out at <math>4^o</math>C and the cells should be kept on ice wherever possible'''
#Centrifuge for 10 minutes at 3000 rpm and <math>4^o</math>C.
#Centrifuge for 10 minutes at 3000 rpm and <math>4^o</math>C.
#Remove supernatant and replace with 10% original volume [[TSS]].
#Remove supernatant. The cell pellets should be sufficiently solid that you can just pour off the supernatant if you are careful.
#*Note: Qualitative experience shows better results if aliquots are made in the <math>4^o</math>C room.
#Resuspend in chilled [[TSS]] buffer. The volume of [[TSS]] to use is 10% of the culture volume that you spun down.
#*Note: It is best if you keep the cells on ice once they are resuspended in TSS and keep tubes that you aliquot them into on ice.[[User:Kathmc|Kathleen]]
#Using a repeating pipettor, add 100 &mu;l aliquots to your chilled eppendorfs and store at <math>-80^o</math>C.
#Make 100 &mu;l aliquots and store at <math>-80^o</math>C.
 
==Use==
#Thaw TSS cells on ice.
#Add DNA, pipette gently to mix (1&mu;l of prepped plasmid is more than enough).
#*Note: If you are adding small volumes (~1&mu;l), be careful to mix the culture well. Diluting the plasmid back into a larger volume can also help.
#Let sit for 30 minutes on ice.
#*Note: If you are in a rush, you can shorten this incubation time to 5-10 min.
#Incubate cells for 30 seconds at <math>42^o</math>C.
#*Note: According to the [http://www.pubmedcentral.gov/articlerender.fcgi?tool=pubmed&pubmedid=2648393 original TSS paper] and qualitative experience ([[Josh Michener|JM]]), this step is completely optional and may actually reduce transformation efficiency.
#Add 1 mL [[SOC]] ([[2XYT]] and LB are also suitable) at room temp.
#Incubate for 1 hour at <math>37^o</math>C on shaker.
#*Note: Can also save some time here by reducing incubation to ~45 min.
#Spread 100-300 &mu;l onto a plate made with appropriate antibiotic.
#Grow overnight at 37 &deg;C.
#Save the rest of the transformants in liquid culture at 4 &deg;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.


==Buffers==
==Buffers==

Revision as of 10:07, 16 January 2006

Materials

  • Plate of cells to be made competent
  • TSS buffer
  • LB media
  • Ice

==Glassware & Equipment

  • Falcon tubes
  • 500μl Eppendorf tubes, on ice
  • 5ml pipete
  • 200ml conical flask
  • Culture tube
  • Repeating pipettor, tip that can make 100μl aliquots.

Preparation

  1. Grow a 5ml overnight culture of cells in LB media using a culture tube. In the morning, dilute this culture back into 25-50ml of fresh LB media in a 200ml conical flask. You should aim to dilute the overnight culture by at least 1/500.
  2. Grow the culture to an OD600 of 0.2 - 0.5. (You will get a very small pellet if you grow 25ml to OD600 0.2)
  3. Put eppendorf tubes on ice now so that they are cold when cells are aliquoted into them later. If your culture is Xml, you will need X tubes. At this point you should also make sure that your TSS is being chilled (it should be stored at 4C but if you have just made it fresh then put it in an ice bath).
  4. Split the culture into two 50ml falcon tubes.

All subsequent steps should be carried out at [math]\displaystyle{ 4^o }[/math]C and the cells should be kept on ice wherever possible

  1. Centrifuge for 10 minutes at 3000 rpm and [math]\displaystyle{ 4^o }[/math]C.
  2. Remove supernatant. The cell pellets should be sufficiently solid that you can just pour off the supernatant if you are careful.
  3. Resuspend in chilled TSS buffer. The volume of TSS to use is 10% of the culture volume that you spun down.
  4. Using a repeating pipettor, add 100 μl aliquots to your chilled eppendorfs and store at [math]\displaystyle{ -80^o }[/math]C.

Buffers

TSS (50 mL)

  • 5g PEG 8000
  • 1.5 mL 1M MgCl2
  • 2.5 mL DMSO
  • LB to 50 mL

Filter sterilize (0.22 μm filter) and store at 4˚C or -20˚C.

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