Haynes:TransformationPlasmids

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Current revision (19:48, 4 March 2013) (view source)
(Traditional Method: Chemically competent cells + ligation)
 
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<- [[Haynes:Protocols | Back to Protocols]]
<- [[Haynes:Protocols | Back to Protocols]]
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=Chemically competent cells + plasmid miniprep=
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=Traditional Method: Chemically competent cells + ligation=
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<div style="width: 800px">
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This method is useful for increasing the efficiency of plasmid up-take.
 +
 
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# Incubate chemically competent cells on ice just until thawed. You will need 30 μL cells per ligation.
 +
# Add 30 μL thawed cells to to 1 sterile 2.0 mL tube (per ligation).
 +
# Add the total ligation reaction to the cells in each 2.0 mL tube. Pipette up and down '''gently''' to mix the cells and DNA.
 +
# Incubate on ice for 10 min.
 +
# Heat shock: Transfer the tubes to 42°C for exactly 45 seconds (heat shock) on a heat block or water bath, then immediately place the tubes on ice for 1 minute.
 +
# Add 750 μL sterile SOC medium to each sample.
 +
# Recovery: Close the caps tightly. Place the tubes in the shaking incubator, secured in a sideways position with tape. Incubate the tubes, with shaking, at 37°C for 45 minutes.
 +
# Pre-warm the agar plates: Incuabte the selection agar plates (one per sample) at 37°C during the 45 minute recovery period.
 +
# Pellet the cells by centrifugation at top speed for 3 minutes.
 +
# Discard the SOC supernatant.
 +
# Resuspend the pellet in 100 μL LB medium (plus proper antibiotic).
 +
# Pipette the total volume of cells onto the agar; spread using sterile glass beads.
 +
# Incubate the inverted plate(s) overnight at 37°C to get colonies.
 +
Note: To store the colonies long term, seal the plate with parafilm and keep the plate at 4°C (inverted).
 +
</div>
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<br><br>
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=Quick Method: Chemically competent cells + plasmid mini prep=
<div style="width: 800px">
<div style="width: 800px">
# Warm selection agar plates at 37°C (one for each plasmid, plus one for a zero plasmid control) for at least 15 min.
# Warm selection agar plates at 37°C (one for each plasmid, plus one for a zero plasmid control) for at least 15 min.
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# Incubate DH5α Turbo competent cells on ice just until thawed. You will need 30 μL cells per ligation.
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# Incubate DH5α Turbo competent cells on ice just until thawed. You will need 30 μL cells per plasmid sample.
# Dilute 0.5 μL plasmid DNA (concentration not important) in 10 μL sterile dH<sub>2</sub>O in sterile 0.5 mL tubes.
# Dilute 0.5 μL plasmid DNA (concentration not important) in 10 μL sterile dH<sub>2</sub>O in sterile 0.5 mL tubes.
# Make a "negative control" sample by simply putting 10 μL sterile dH<sub>2</sub>O in a tube without DNA.
# Make a "negative control" sample by simply putting 10 μL sterile dH<sub>2</sub>O in a tube without DNA.
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# Pipette the total volume of cells + DNA onto the agar; spread using sterile glass beads.
# Pipette the total volume of cells + DNA onto the agar; spread using sterile glass beads.
# Incubate the inverted plate(s) overnight at 37°C to get colonies. Seal the plate with parafilm and store the plate at 4°C (inverted).
# Incubate the inverted plate(s) overnight at 37°C to get colonies. Seal the plate with parafilm and store the plate at 4°C (inverted).
-
Note: The negative control will show you the number of “background” colonies so that you can determine whether your transformation worked, or is just the result of selection failure.
+
 
 +
Note: The negative control will show you the number of “background” colonies so that you can determine whether your transformation worked, or is just the result of selection failure.<br>
 +
Note 2: This method can be used for ligations, where construction is very straight-forward and your DNA material is highly concentrated. It works well for "forced" ligations (sticky overhangs, no unwanted annealing)
</div>
</div>
 +
<br><br>
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=Chemically competent cells + iGEM Registry sample=
+
=Quick Method: Chemically competent cells + iGEM Registry sample=
<div style="width: 800px">
<div style="width: 800px">
# Warm selection agar plates at 37°C (one for each plasmid, plus one for a zero plasmid control) for at least 15 min.
# Warm selection agar plates at 37°C (one for each plasmid, plus one for a zero plasmid control) for at least 15 min.
-
# Incubate DH5α Turbo competent cells on ice just until thawed. You will need 30 μL cells per ligation.
+
# Incubate DH5α Turbo competent cells on ice just until thawed. You will need 30 μL cells per plasmid sample.
# <font color="blue">Locate the desired well in the Registry plate. Inject 10 μL sterile dH<sub>2</sub>O into the well and pipette up and down to resuspend the dried DNA and indicator dye. Transfer each DNA solution to a  sterile 0.5 mL tube.</font>
# <font color="blue">Locate the desired well in the Registry plate. Inject 10 μL sterile dH<sub>2</sub>O into the well and pipette up and down to resuspend the dried DNA and indicator dye. Transfer each DNA solution to a  sterile 0.5 mL tube.</font>
# Make a "negative control" sample by simply putting 10 μL sterile dH<sub>2</sub>O in a tube without DNA.
# Make a "negative control" sample by simply putting 10 μL sterile dH<sub>2</sub>O in a tube without DNA.
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# Pipette the total volume of cells + DNA onto the agar; spread using sterile glass beads.
# Pipette the total volume of cells + DNA onto the agar; spread using sterile glass beads.
# Incubate the inverted plate(s) overnight at 37°C to get colonies. Seal the plate with parafilm and store the plate at 4°C (inverted).
# Incubate the inverted plate(s) overnight at 37°C to get colonies. Seal the plate with parafilm and store the plate at 4°C (inverted).
 +
Note: The negative control will show you the number of “background” colonies so that you can determine whether your transformation worked, or is just the result of selection failure.
Note: The negative control will show you the number of “background” colonies so that you can determine whether your transformation worked, or is just the result of selection failure.
</div>
</div>

Current revision

<- Back to Protocols

Traditional Method: Chemically competent cells + ligation

This method is useful for increasing the efficiency of plasmid up-take.

  1. Incubate chemically competent cells on ice just until thawed. You will need 30 μL cells per ligation.
  2. Add 30 μL thawed cells to to 1 sterile 2.0 mL tube (per ligation).
  3. Add the total ligation reaction to the cells in each 2.0 mL tube. Pipette up and down gently to mix the cells and DNA.
  4. Incubate on ice for 10 min.
  5. Heat shock: Transfer the tubes to 42°C for exactly 45 seconds (heat shock) on a heat block or water bath, then immediately place the tubes on ice for 1 minute.
  6. Add 750 μL sterile SOC medium to each sample.
  7. Recovery: Close the caps tightly. Place the tubes in the shaking incubator, secured in a sideways position with tape. Incubate the tubes, with shaking, at 37°C for 45 minutes.
  8. Pre-warm the agar plates: Incuabte the selection agar plates (one per sample) at 37°C during the 45 minute recovery period.
  9. Pellet the cells by centrifugation at top speed for 3 minutes.
  10. Discard the SOC supernatant.
  11. Resuspend the pellet in 100 μL LB medium (plus proper antibiotic).
  12. Pipette the total volume of cells onto the agar; spread using sterile glass beads.
  13. Incubate the inverted plate(s) overnight at 37°C to get colonies.

Note: To store the colonies long term, seal the plate with parafilm and keep the plate at 4°C (inverted).



Quick Method: Chemically competent cells + plasmid mini prep

  1. Warm selection agar plates at 37°C (one for each plasmid, plus one for a zero plasmid control) for at least 15 min.
  2. Incubate DH5α Turbo competent cells on ice just until thawed. You will need 30 μL cells per plasmid sample.
  3. Dilute 0.5 μL plasmid DNA (concentration not important) in 10 μL sterile dH2O in sterile 0.5 mL tubes.
  4. Make a "negative control" sample by simply putting 10 μL sterile dH2O in a tube without DNA.
  5. Add 30 μL thawed cells each tube of diluted DNA (and the negative control). Immediately place on ice and incubate for 10 min. (Do not heat shock; No 30 min. recovery is required for Amp or Kan resistance)
  6. Label the pre-warmed plates with the antibiotic name, strain name, DNA name, your initials, and the date.
  7. Pipette the total volume of cells + DNA onto the agar; spread using sterile glass beads.
  8. Incubate the inverted plate(s) overnight at 37°C to get colonies. Seal the plate with parafilm and store the plate at 4°C (inverted).

Note: The negative control will show you the number of “background” colonies so that you can determine whether your transformation worked, or is just the result of selection failure.
Note 2: This method can be used for ligations, where construction is very straight-forward and your DNA material is highly concentrated. It works well for "forced" ligations (sticky overhangs, no unwanted annealing)



Quick Method: Chemically competent cells + iGEM Registry sample

  1. Warm selection agar plates at 37°C (one for each plasmid, plus one for a zero plasmid control) for at least 15 min.
  2. Incubate DH5α Turbo competent cells on ice just until thawed. You will need 30 μL cells per plasmid sample.
  3. Locate the desired well in the Registry plate. Inject 10 μL sterile dH2O into the well and pipette up and down to resuspend the dried DNA and indicator dye. Transfer each DNA solution to a sterile 0.5 mL tube.
  4. Make a "negative control" sample by simply putting 10 μL sterile dH2O in a tube without DNA.
  5. Add 30 μL thawed cells each tube of diluted DNA (and the negative control). Immediately place on ice and incubate for 10 min. (Do not heat shock; No 30 min. recovery is required for Amp or Kan resistance)
  6. Label the pre-warmed plates with the antibiotic name, strain name, DNA name, your initials, and the date.
  7. Pipette the total volume of cells + DNA onto the agar; spread using sterile glass beads.
  8. Incubate the inverted plate(s) overnight at 37°C to get colonies. Seal the plate with parafilm and store the plate at 4°C (inverted).

Note: The negative control will show you the number of “background” colonies so that you can determine whether your transformation worked, or is just the result of selection failure.

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