# Preparing chemically competent cells

(Difference between revisions)
 Revision as of 09:37, 26 May 2005 (view source)← Previous diff Revision as of 21:39, 12 April 2010 (view source)m Next diff → (46 intermediate revisions not shown.) Line 1: Line 1: - *Grow a 5-25mL culture to an OD600 of 0.2 $\rightarrow$ 0.5 + {{back to protocols}} - *Centrifuge for 10 minutes at 3000rpm and $4^o$C. + ==Materials== - *Remove supernatant and replace with 10% original volume TSS. + *Plate of cells to be made competent - *Make 100uL aliquots and store at $-80^o$C. + *[[TSS|TSS buffer]] + *[[LB|LB media]] + *Ice + ==Glassware & Equipment== + *Falcon tubes + *500μl Eppendorf tubes, on ice + *200ml conical flask + *200μl pipetman or repeating pipettor + *5ml pipette + ==Preparation== + #Grow a 5ml [[Bacterial cell culture|overnight culture]] of cells in LB media.  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 1/100. + #Grow the diluted culture to an OD600 of 0.2 - 0.5. (You will get a very small pellet if you grow 25ml to OD600 0.2) + #Put eppendorf tubes on ice now so that they are cold when cells are aliquoted into them later.  If your culture is X ml, you will need X tubes.  At this point you should also make sure that your [[TSS]] is being chilled (it should be stored at $4^o$C but if you have just made it fresh then put it in an ice bath). + #Split the culture into two 50ml falcon tubes and incubate on ice for 10 min. + '''All subsequent steps should be carried out at $4^o$C and the cells should be kept on ice wherever possible''' + #Centrifuge for 10 minutes at 3000 rpm and $4^o$C. + #Remove supernatant.  The cell pellets should be sufficiently solid that you can just pour off the supernatant if you are careful.  Pipette out any remaining media. + #Resuspend in chilled [[TSS]] buffer.  The volume of [[TSS]] to use is 10% of the culture volume that you spun down.  You may need to vortex gently to fully resuspend the culture, keep an eye out for small cell aggregates even after the pellet is completely off the wall. + #Add 100 μl aliquots to your chilled eppendorfs and store at $-80^o$C. + #*The original paper chung suggests freezing the cells immediately using a dry ice bath.  I ([[Barry Canton|BC]]) have used liquid nitrogen quite successfully instead of dry ice. Simply placing the cells at $-80^o$C also seems to work well ([[User:Jkm|Jkm]]) + #*If you run a control every time you clone (i.e. a vector-only ligation), you can as well freeze cells in 200 μl aliquots. Unused cells can be frozen back once and reused, albeit with some loss of competence. + #It is a good idea to run a positive control on the cells. + #*The [[Endy Lab]] is trying to use a standard positive control to better compare (and hopefully improve) the transformation efficiencies in the lab, you can check it out [[Endy:Standard transformation positive control|here]]. + ==Related topics & References== + *[[TSS|Preparing TSS buffer]] + *[[Transforming chemically competent cells]]'' + *[[Electrocompetent cells|Preparing electrocompetent cells]] + *[[Electroporation]] + *[[Bacterial cell culture]] - [[TSS]] (50 mL) + Based on a protocol from [[Kathleen McGinness]], annotated by [[Josh Michener]] & [[Barry Canton]]. Original protocol published by Chung et al.chung chung2 - *5g PEG 8000 + - *1.5 mL 1M MgCl2 + - *2.5 mL DMSO + - *LB to 50 mL + - [[Category:E.Coli Protocol]] + + #chung pmid=2648393 + #chung2 pmid=8510550 + + + [[Category:Protocol]] + [[Category:In vivo]] + [[Category:Escherichia coli]]

## Glassware & Equipment

• Falcon tubes
• 500μl Eppendorf tubes, on ice
• 200μl pipetman or repeating pipettor
• 5ml pipette

## Preparation

1. Grow a 5ml overnight culture of cells in LB media. 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 1/100.
2. Grow the diluted 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 X ml, you will need X tubes. At this point you should also make sure that your TSS is being chilled (it should be stored at 4oC but if you have just made it fresh then put it in an ice bath).
4. Split the culture into two 50ml falcon tubes and incubate on ice for 10 min.

All subsequent steps should be carried out at 4oC and the cells should be kept on ice wherever possible

1. Centrifuge for 10 minutes at 3000 rpm and 4oC.
2. Remove supernatant. The cell pellets should be sufficiently solid that you can just pour off the supernatant if you are careful. Pipette out any remaining media.
3. Resuspend in chilled TSS buffer. The volume of TSS to use is 10% of the culture volume that you spun down. You may need to vortex gently to fully resuspend the culture, keep an eye out for small cell aggregates even after the pellet is completely off the wall.
4. Add 100 μl aliquots to your chilled eppendorfs and store at − 80oC.
• The original paper [1] suggests freezing the cells immediately using a dry ice bath. I (BC) have used liquid nitrogen quite successfully instead of dry ice. Simply placing the cells at − 80oC also seems to work well (Jkm)
• If you run a control every time you clone (i.e. a vector-only ligation), you can as well freeze cells in 200 μl aliquots. Unused cells can be frozen back once and reused, albeit with some loss of competence.
5. It is a good idea to run a positive control on the cells.
• The Endy Lab is trying to use a standard positive control to better compare (and hopefully improve) the transformation efficiencies in the lab, you can check it out here.

## Related topics & References

Based on a protocol from Kathleen McGinness, annotated by Josh Michener & Barry Canton. Original protocol published by Chung et al.[1] [2]

1. Chung CT, Niemela SL, and Miller RH. . pmid:2648393. [chung]
2. Chung CT and Miller RH. . pmid:8510550. [chung2]
All Medline abstracts: PubMed HubMed