Electrocompetent cells

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[[Category: E.Coli Protocol]]
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{{back to protocols}}
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==Specific protocols==
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[[Knight:Preparing electrocompetent cells]]
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[[Belcher/Knight: Electrocompetent Cells]]
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[[Richard Lab:Preparing electrocompetent cells]]
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see also [[Electroporation]]
==Growing Electrocompetent Cells==
==Growing Electrocompetent Cells==
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===Materials===
===Materials===
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GYT<br>
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[[GYT]]  (glycerol, yeast extract, tryptone)<br>
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DI water<br>
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:• 10%(v/v) glycerol
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:• 0.125% (w/v) yeast extract
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:• 0.25% (w/v) tryptone
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DI water<br>
10% Glycerol<br>
10% Glycerol<br>
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===Special Equipment===
===Special Equipment===
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*Dilute and incubate: Inoculate two aliquots of 475 ml of prewarmed LB medium in separate 2-liter flasks with 25 ml of the overnight bacterial culture. Incubate the flasks at 37&deg;C with agitation (300 cycles/min in a rotary shaker). Measure the OD-600 every twenty minutes (this step will take around 1.5-2 hrs).
*Dilute and incubate: Inoculate two aliquots of 475 ml of prewarmed LB medium in separate 2-liter flasks with 25 ml of the overnight bacterial culture. Incubate the flasks at 37&deg;C with agitation (300 cycles/min in a rotary shaker). Measure the OD-600 every twenty minutes (this step will take around 1.5-2 hrs).
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*Rapidly cool culture: Once the OD-600 of the culture reaches 0.6-1.0, rapidly transfer the flasks to the pre-made ice-water bath for 15-30 minutes. Swirl the culture occasionally to ensure that cooling occurs evenly.  In preparation for the next step, place the centrifuge bottles in the ice-water bath as well.
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*Rapidly cool culture: Once the OD-600 of the culture reaches 0.6-1.0 ([[Molecular Cloning]] recommends 0.4), rapidly transfer the flasks to the pre-made ice-water bath for 15-30 minutes. Swirl the culture occasionally to ensure that cooling occurs evenly.  In preparation for the next step, place the centrifuge bottles in the ice-water bath as well.
''Note: After this point, do not let your cells warm up past 4&deg;C''
''Note: After this point, do not let your cells warm up past 4&deg;C''
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''Note: When harvesting cells by decanting, be very careful to not disturb the pellet-- this could result in a much lower yield. If necessary, aspirate instead of decant the supernatant. Get someone to show you how to aspirate.  Also, if the pellet seems loose, sometimes it is helpful to re-spin the cells down.''
''Note: When harvesting cells by decanting, be very careful to not disturb the pellet-- this could result in a much lower yield. If necessary, aspirate instead of decant the supernatant. Get someone to show you how to aspirate.  Also, if the pellet seems loose, sometimes it is helpful to re-spin the cells down.''
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*Centrifuge 1: Transfer the cultures to ice-cold centrifuge bottles.  Harvest the cells by centrifugation at 1000g (2500 rpm) for 15 minutes at 4&deg;C. Decant the supernantant and resuspend the cell pellet in 500 ml of ice-cold DI water.
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*Centrifuge 1: Transfer the cultures to ice-cold centrifuge bottles.  Harvest the cells by centrifugation at 1000g (2500 rpm) for 15 minutes at 4&deg;C. Decant the supernantant and resuspend the cell pellet in 500 ml of ice-cold DI water.  ''Note: I think this should be done for each of the two 500ml cultures, i.e this is a 1:1 resuspension rather than a concentration by a factor of 2 [[Barry Canton|BC]]''.
*Centrifuge 2 (water): Harvest the cells by centrifugation at 1000g for 20 minutes at 4&deg;C. Decant the supernatant and resuspend the cell pellet in 250 ml ice-cold DI water.
*Centrifuge 2 (water): Harvest the cells by centrifugation at 1000g for 20 minutes at 4&deg;C. Decant the supernatant and resuspend the cell pellet in 250 ml ice-cold DI water.
*Centrifuge 3 (water): Harvest the cells by centrifugation at 1000g for 20 minutes at 4&deg;C. Decant the supernatant and resuspend the cell pellet in 10 ml ice-cold 10% glycerol.
*Centrifuge 3 (water): Harvest the cells by centrifugation at 1000g for 20 minutes at 4&deg;C. Decant the supernatant and resuspend the cell pellet in 10 ml ice-cold 10% glycerol.
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**''To spin down your pellet in 10 ml, it might be helpful to use 15-ml Falcon tubes instead of the round-bottom tubes. [[Kelly Chang|KC]]''
*Centrifuge 4 (glycerol): Harvest the cells by centrifugation at 1000g for 20 minutes at 4&deg;C. Carefully decant the supernatant and use a Pastteur pipette attached to a vacuum line to remove any remaining drops of buffer.
*Centrifuge 4 (glycerol): Harvest the cells by centrifugation at 1000g for 20 minutes at 4&deg;C. Carefully decant the supernatant and use a Pastteur pipette attached to a vacuum line to remove any remaining drops of buffer.
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*Resuspend in GYT: Resuspend in 1 ml ice cold GYT. This is best done by gently swirling rather pipetting or vortexing.
*Resuspend in GYT: Resuspend in 1 ml ice cold GYT. This is best done by gently swirling rather pipetting or vortexing.
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*Measure OD: Measure the OD-600 of a 1:100 dilution of the cell suspension. (In the cuvette, mix 0.99 <math>\mu</math>L water and 0.01 <math>\mu</math>L cell suspensiono).
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*Measure OD: Measure the OD-600 of a 1:100 dilution of the cell suspension. (In the cuvette, mix 0.99 mL water and 0.01 mL cell suspension).
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''Note: The desired concentration is <math>2.5 \times 10^{11}</math> cells per mL, giving <math>1\times 10^{10}</math> cells per 40 <math>\mu</math>L. This corresponds to an OD-600 (after 100x dilution) of roughly 3.75. It is difficult to reach this value, but it is still important to know the concentration of cells to calculate efficiencies.
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''Note: The desired concentration is <math>2.5 \times 10^{11}</math> cells per mL, giving <math>1\times 10^{10}</math> cells per 40 <math>\mu</math>L. This corresponds to an OD-600 (after 100x dilution) of roughly 3.75. It is difficult to reach this value, but it is still important to know the concentration of cells to calculate efficiencies.
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* Dilute the cell suspension to a concentration of 2 x 10^10 to 3 x 10^10 cells/ml (1.0 OD600 = approx. 2.5 x 10^8 cells/ml) with ice-cold GYT medium.  
*Test for arcing: Transfer 40 ul of the suspension to an ice-cold electroporation cuvette (0.1-0.2 cm gap, on middle shelf next to electroporator) and test whether arcing occurs when an electrical discharge is applied. Place the cuvette in the green holder attached to the machine. Go to option 4, Pre-set protocols; choose bacterial; choose the correct choice for your size cuvette, probably the first option for a .1 cm cuvette. If arcing occurs, wash the remainder of the cell suspension once more with ice-cold GYT medium to ensure that the conductivity of the bacterial suspension is sufficiently low (<5 mEq).
*Test for arcing: Transfer 40 ul of the suspension to an ice-cold electroporation cuvette (0.1-0.2 cm gap, on middle shelf next to electroporator) and test whether arcing occurs when an electrical discharge is applied. Place the cuvette in the green holder attached to the machine. Go to option 4, Pre-set protocols; choose bacterial; choose the correct choice for your size cuvette, probably the first option for a .1 cm cuvette. If arcing occurs, wash the remainder of the cell suspension once more with ice-cold GYT medium to ensure that the conductivity of the bacterial suspension is sufficiently low (<5 mEq).
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To use frozen cells: Remove an appropriate number of aliquots of cells from the -80&deg;C freezer. Thaw the tubes on ice.
To use frozen cells: Remove an appropriate number of aliquots of cells from the -80&deg;C freezer. Thaw the tubes on ice.
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==Preparing electrically competent ''E. coli'' cells==
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==Notes==
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*'''[[User:Jason R. Kelly|Jason R. Kelly]] 15:14, 13 July 2007 (EDT):''' You might consider using a syringe filter rather than spins to do the washes when preparing the cells.  This can apparently save a lot of time, though I've yet to try it myself.  
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Protocol from Austin Che and annotated by Reshma Shetty based on online research.<br>
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*'''[[User:Hugh Kingston|hugh kingston]] 08:09, 17 April 2008 (EDT)''': I tried with a 0.22μm syringe filter and 50ml OD 600 e.coli. The filter kept jamming, not very successful. Perhaps a larger, vacuum filter would be better?
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[[Category:Protocol]]
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===Materials===
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[[Category:In vivo]]
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[[Category:Escherichia coli]]
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====Equipment====
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*-80&deg;C freezer
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*37&deg;C incubator
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*Refridgerated centrifuge that accepts 225 mL culture tubes
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*Refridgerated centrifuge that accepts 1.5 mL tubes
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====Chemicals and reagents====
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*~500 mL LB Lennox supplemented with appropriate concentration of antibiotic if appropriate.
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*~600 mL sterile deionized water chilled to 4&deg;C
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*~22 mL sterile 15% glycerol in deionized water chilled to 4&deg;C
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*Ice bucket and ice
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*Dry ice, ethanol bath
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====Supplies====
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*Many 0.6 mL plastic tubes chilled to -80 &deg;C
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*14 mL culture tube for starter culture
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*2 L flask for culture
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*225 mL plastic tubes for centrifugation
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*Pipets
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*Sucking pipet
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===Procedure===
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#Prechill all tubes and pipets at 4&deg;C or -80&deg;C as appropriate.  <br> Also rinse all flasks with H<sub>2</sub>O prior to autoclaving in order to remove residual detergents that may remain on glassware from dishwashing.  This step may increase competency.
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#Inoculate 5mL LB medium and grow overnight at 37&deg;C with rotation. <br> Use LB Lennox rather than LB Miller in order to lessen salt content of media
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# Add the 5mL overnight culture to 450mL LB medium and incubate at 37&deg;C with vigorous shaking until the OD 600nm is between 0.5 and 1.0It should take about 3 hours. <br> For recA<sup>-</sup> strains, the OD 600 nm should be between 0.5 and 0.7 according to one online source.
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# Pour the culture into two 225 mL centrifuge tubes.
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# Place the tubes on ice for 15 minutes.  <br> This step can vary in incubation time between 15 minutes and 1 hr.  Longer incubation times may lead to higher competency.<br>''For the following steps it is important to keep cells cold and remove all the supernatant in each step to remove residual ions.''<br>
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# Centrifuge for 10 mins at 2000g at 4&deg;C
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# Remove supernatant and gently resuspend pellets with 200mL cold sterile water. <br> Initially add 10-20 mL of water and resuspend by pipetting. Then add the rest of the water.
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# Centrifuge for 15 mins at 2000g at 4&deg;C
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# Remove supernatant and gently resuspend pellets with 100mL cold sterile water. <br> Initially add 10-20 mL of water and resuspend by pipetting.  Then add the rest of the water.
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# Centrifuge for 15 mins at 2000g at 4&deg;C
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# Remove supernatant and gently resuspend pellets with 10mL cold 15% glycerol.  <br> This can be optionally transferred to a 14 mL conical tube.
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# Centrifuge for 15 mins at 1500g at 4&deg;C
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# Remove supernatant and transfer pellet to 1.5 mL tube using sucking pipet.  <br> There should be sufficient glycerol in the pellet for you to do this.
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# Microcentrifuge 5 mins at maximum speed at 4&deg;C.
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# Remove supernatant.
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# Resuspend pellet in 1mL cold 15% glycerol.
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# Aliquot 50 &mu;L per tube.
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# Shock freeze cell suspensions in a dry ice and ethanol bath.  <br> One website recommended against using liquid nitrogen but did not justify this recommendation.
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# Store at -80&deg;C
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Current revision

back to protocols

Contents

Specific protocols

Knight:Preparing electrocompetent cells

Belcher/Knight: Electrocompetent Cells

Richard Lab:Preparing electrocompetent cells

see also Electroporation

Growing Electrocompetent Cells

Copied and edited from matterhorn.lcs.mit.edu/biosmug; originally from Sambrook and Russell's "Molecular Cloning: A Laboratory Manual" Third Edition.

(Note: once the cells are grown and have been placed in the first ice bath, you do not want the temperature of the sample to rise above 4 °C at any point. Therefore, many of the instructions are given with this in mind; always think ahead to the next step and to how you are going to keep your cells from warming up. This includes prechilling tubes and keeping all wash materials and samples on ice.)

Materials

GYT (glycerol, yeast extract, tryptone)

• 10%(v/v) glycerol
• 0.125% (w/v) yeast extract
• 0.25% (w/v) tryptone

DI water
10% Glycerol


Special Equipment

Centrifuge
Ice water bath
Liquid nitrogen

Method

Important: All steps in this protocol should be carried out aseptically

  • Inoculate: Prepare flask containing 50 ml of LB medium. Pick up a single colony of cells from plate (using a sterile toothpick) and swirl around inside flask. Incubate the culture overnight at 37°C with vigorous aeration (250 pm in a rotary shaker).
  • Dilute and incubate: Inoculate two aliquots of 475 ml of prewarmed LB medium in separate 2-liter flasks with 25 ml of the overnight bacterial culture. Incubate the flasks at 37°C with agitation (300 cycles/min in a rotary shaker). Measure the OD-600 every twenty minutes (this step will take around 1.5-2 hrs).
  • Rapidly cool culture: Once the OD-600 of the culture reaches 0.6-1.0 (Molecular Cloning recommends 0.4), rapidly transfer the flasks to the pre-made ice-water bath for 15-30 minutes. Swirl the culture occasionally to ensure that cooling occurs evenly. In preparation for the next step, place the centrifuge bottles in the ice-water bath as well.

Note: After this point, do not let your cells warm up past 4°C

Note: When harvesting cells by decanting, be very careful to not disturb the pellet-- this could result in a much lower yield. If necessary, aspirate instead of decant the supernatant. Get someone to show you how to aspirate. Also, if the pellet seems loose, sometimes it is helpful to re-spin the cells down.

  • Centrifuge 1: Transfer the cultures to ice-cold centrifuge bottles. Harvest the cells by centrifugation at 1000g (2500 rpm) for 15 minutes at 4°C. Decant the supernantant and resuspend the cell pellet in 500 ml of ice-cold DI water. Note: I think this should be done for each of the two 500ml cultures, i.e this is a 1:1 resuspension rather than a concentration by a factor of 2 BC.
  • Centrifuge 2 (water): Harvest the cells by centrifugation at 1000g for 20 minutes at 4°C. Decant the supernatant and resuspend the cell pellet in 250 ml ice-cold DI water.
  • Centrifuge 3 (water): Harvest the cells by centrifugation at 1000g for 20 minutes at 4°C. Decant the supernatant and resuspend the cell pellet in 10 ml ice-cold 10% glycerol.
    • To spin down your pellet in 10 ml, it might be helpful to use 15-ml Falcon tubes instead of the round-bottom tubes. KC
  • Centrifuge 4 (glycerol): Harvest the cells by centrifugation at 1000g for 20 minutes at 4°C. Carefully decant the supernatant and use a Pastteur pipette attached to a vacuum line to remove any remaining drops of buffer.
  • Resuspend in GYT: Resuspend in 1 ml ice cold GYT. This is best done by gently swirling rather pipetting or vortexing.
  • Measure OD: Measure the OD-600 of a 1:100 dilution of the cell suspension. (In the cuvette, mix 0.99 mL water and 0.01 mL cell suspension).

Note: The desired concentration is 2.5 \times 10^{11} cells per mL, giving 1\times 10^{10} cells per 40 μL. This corresponds to an OD-600 (after 100x dilution) of roughly 3.75. It is difficult to reach this value, but it is still important to know the concentration of cells to calculate efficiencies.

  • Dilute the cell suspension to a concentration of 2 x 10^10 to 3 x 10^10 cells/ml (1.0 OD600 = approx. 2.5 x 10^8 cells/ml) with ice-cold GYT medium.
  • Test for arcing: Transfer 40 ul of the suspension to an ice-cold electroporation cuvette (0.1-0.2 cm gap, on middle shelf next to electroporator) and test whether arcing occurs when an electrical discharge is applied. Place the cuvette in the green holder attached to the machine. Go to option 4, Pre-set protocols; choose bacterial; choose the correct choice for your size cuvette, probably the first option for a .1 cm cuvette. If arcing occurs, wash the remainder of the cell suspension once more with ice-cold GYT medium to ensure that the conductivity of the bacterial suspension is sufficiently low (<5 mEq).
  • Storage: Store cells at -80°C until they are required for use. For storage, dispense 40 ul aliquots of the cell suspension into sterile, ice-cold .5 ml microcentrifuge tubes, drop into a bath of liquid nitrogen and transfer to a -80°C freezer. To remove the tubes from the liquid nitrogen bath, bring out into the hall along with a storage box, and pour the tubes and liquid nitrogen into the box. Once all the tubes are out, close the box most of theh way and let the liquid run out into the hallway. Try not to do this in the very center of the walkway!

To use frozen cells: Remove an appropriate number of aliquots of cells from the -80°C freezer. Thaw the tubes on ice.

Notes

  • Jason R. Kelly 15:14, 13 July 2007 (EDT): You might consider using a syringe filter rather than spins to do the washes when preparing the cells. This can apparently save a lot of time, though I've yet to try it myself.
  • hugh kingston 08:09, 17 April 2008 (EDT): I tried with a 0.22μm syringe filter and 50ml OD 600 e.coli. The filter kept jamming, not very successful. Perhaps a larger, vacuum filter would be better?
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