Difference between revisions of "Help:Protocols/Transformation"

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=Transformation Protocol=
 
=Transformation Protocol=
−
'''Estimated time: 3 hours''' (plus 14-18 hour incubation)<br>
+
[https://static.igem.org/mediawiki/parts/6/67/IGEM_Registry_-_Transformation_Protocol.pdf Printable PDF Protocol]<br />
 +
'''Estimated bench time: 1 hour'''<br />
 +
'''Estimated total time: 3 hours''' (plus 14-18 hour incubation)<br />
  
 
Transformations are essential to using the [[Help:DNA_Distribution|DNA Distribution Kits]]: resuspend the DNA sample in a well, transform the DNA into competent cells, and select single colonies. However, transformations can also be one of the more fickle laboratory techniques.  
 
Transformations are essential to using the [[Help:DNA_Distribution|DNA Distribution Kits]]: resuspend the DNA sample in a well, transform the DNA into competent cells, and select single colonies. However, transformations can also be one of the more fickle laboratory techniques.  
  
  
−
At iGEM HQ, we run test transformations of the DNA Distribution Kit with the following protocol. We have found that it is the best protocol to use with Registry samples and ensures high efficiency transformations. This protocol may be particularly useful if you are finding that your transformations are not working, or yielding few colonies.
+
At iGEM HQ, we run test transformations of the DNA Distribution Kit with the following protocol. We have found that it is the best protocol to use with the DNA Distribution Kit and ensures high efficiency transformations.
  
  
−
==Important Reminders==
+
*At iGEM HQ, we make our own stocks of [[Help:Protocols/Competent_Cells|NEB 10b competent cells]]. Competent cells purchased from vendors will have better efficiency.
−
*At iGEM HQ, we make our own stocks of [[Help:Protocols/Competent_Cells|NEB 10b competent cells]]. Competent cells purchased from vendors will have better efficiency.  
+
*Make sure to test the competency of your cells with the provided [[Help:Competent_Cell_Test_Kit|Competent Cell Test Kit]].
−
*'''Make sure to test the competency of your cells.''' We provide a [[Help:Competent_Cell_Test_Kit|Competent Cell Test Kit]] to test the competency of your cells with different concentrations of DNA.
+
 
*'''Read through the entire protocol before starting!'''
 
*'''Read through the entire protocol before starting!'''
  
  
 
==Materials==
 
==Materials==
−
*'''Resuspended DNA''' (''Resuspend well with 10ul dH20, pipet up and down several times, let sit for a few minutes. Resuspension will be red, from cresol red dye.'')
+
<html>
−
*'''10pg/ul Control''' (''pSB1C3 w/ BBa_J04450'')
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<table class="protocolTable sortable">
−
*'''[[Help:Protocols/Competent_Cells | Competent cells]]''' (''50ul per transformation. We store competent cells in aliquots of 260ul for a total of 5 reactions'')
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<tr>
−
*'''Ice''' (''in ice bucket/container'')
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<th style="width: 150px;">Material</th>
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*'''2ml tube''' (''1 per a transformation. We recommend labelling tubes before getting started.'')
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<th>Description/Comments</th>
−
*'''42ºC water bath'''
+
</tr>
−
*'''SOC media''' (''check for contamination!'')
+
<tr>
−
*'''Petri dishes with LB agar and appropriate antibiotic''' (''2 per transformation, for a 20ul and 200ul plating'')
+
<td class="material">Resuspended DNA</td>
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*'''sterile glass beads or spreader'''
+
<td class="description">Resuspend DNA Distribution Kit well(s) with 10µl dH20. Pipet up and down several times, let sit for a few minutes. Resuspension will be red from cresol red dye.</td>
−
*'''37ºC incubator'''
+
</tr>
 +
<tr>
 +
<td class="material">10pg/µl Control DNA</td>
 +
<td class="description">1µl for control transformation. pSB1C3 w/ BBa_J04450, RFP on high-copy chloramphenicol resistant plasmid. Located in the Competent Cell Test Kit.</td>
 +
</tr>
 +
<tr>
 +
<td class="material">Competent Cells</td>
 +
<td class="description">50µl per transformation. iGEM HQ stores competent cells in aliquots of 260µl (5rxns total) at -80°C.</td>
 +
</tr>
 +
<tr>
 +
<td class="material">2ml Microtubes</td>
 +
<td class="description">One tube per transformation. Label tubes with part name or well location before starting.</td>
 +
</tr>
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<tr>
 +
<td class="material">Floating Foam Tube Rack</td>
 +
<td class="description">Place 2ml tubes in floating tube rack for better support when working on ice and for the heat shock in the water bath.</td>
 +
</tr>
 +
<tr>
 +
<td class="material">Ice & ice bucket</td>
 +
<td class="description">Fill bucket with ice, and pre-chill 2ml tubes (5min). Thaw competent cell stock on ice (10-15min).</td>
 +
</tr>
 +
<tr>
 +
<td class="material">Lab Timer</td>
 +
<td class="description"></td>
 +
</tr>
 +
<tr>
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<td class="material">42°C water bath</td>
 +
<td class="description">Set water bath to 42°C before starting.</td>
 +
</tr>
 +
<tr>
 +
<td class="material">SOC Media</td>
 +
<td class="description">200µl per transformation. SOC Media is better than LB Media for higher transformation efficiency. SOC Media should not contain antibiotics, and can be easily contaminated.</td>
 +
</tr>
 +
<tr>
 +
<td class="material">37°C incubator</td>
 +
<td class="description">Preferably with a rotor/shaker for 2ml tubes. Incubate petri plates overnight (non-agitated).</td>
 +
</tr>
 +
<tr>
 +
<td class="material">Petri plates w/ LB agar and antibiotic</td>
 +
<td class="description">2 plates per transformation: for 20µl and 200µl platings. Make sure to use appropriate antibiotic. Label with part name or well location before starting.</td>
 +
</tr>
 +
<tr>
 +
<td class="material">Sterile spreader or glass beads</td>
 +
<td class="description">Used to spread transformation across petri plates. Be sure to use sterile technique in between platings.</td>
 +
</tr>
 +
<tr>
 +
<td class="material">Pipettes and Tips</td>
 +
<td class="description">10µl, 20µl, 200µl tips and pipettes recommended</td>
 +
</tr>
 +
</table>
 +
</html>
 +
 
 +
 
 +
 
 +
==Setup & Protocol==
 +
When transforming competent cells, both timing and temperature are very important. Use a lab timer, follow the incubation temperatures closely, and keep materials on ice when required.
 +
 
 +
 
 +
Resuspend DNA in selected wells in the Distribution Kit. Label 2ml tubes with part name or well location. Fill lab ice bucket with ice, and pre-chill 2ml tubes (one tube for each transformation, including your control) in a floating foam tube rack.
 +
 
  
−
==Procedure==
+
# '''Thaw competent cells on ice:''' This may take 10-15min for a 260µl stock. Dispose of unused competent cells. Do not refreeze unused thawed cells, as it will drastically reduce transformation efficiency.
−
# Thaw competent cells on ice. This may take 5-10min for a 260ul stock. Pre-chill empty 2ml tube(s) for each transformation.
+
# '''Pipette 50µl of competent cells into 2ml tube:''' 50µl in a 2ml tube per transformation. Tubes should be labeled, pre-chilled, and in a floating tube rack for support. Keep all tubes on ice. Don’t forget a 2ml tube for your control.
−
# Add 50 µL of thawed competent cells into pre-chilled 2ml tube, and another 50µL into a 2ml tube, labelled for '''your control'''.
+
# '''Pipette 1µl of resuspended DNA into 2ml tube:''' Pipette from well into appropriately labeled tube. Gently pipette up and down a few times. Keep all tubes on ice.
−
# Add 1 - 2 µL of resuspended DNA to the 2ml tube. Pipet up and down a few times, gently. Make sure to keep the competent cells on ice.
+
# '''Pipette 1µl of control DNA into 2ml tube:''' Pipette 1µl of 10pg/µl control into your control transformation. Gently pipette up and down a few times. Keep all tubes on ice.
−
# Add 1 µL of the 10pg/ul Control to your control transformation. Pipet up and down a few times, gently.
+
# '''Close 2ml tubes, incubate on ice for 30min:''' Tubes may be gently agitated/flicked to mix solution, but return to ice immediately.
−
# Close tubes and incubate the cells on ice for 30 minutes.
+
# '''Heat shock tubes at 42°C for 1 min:''' 2ml tubes should be in a floating foam tube rack. Place in water bath to ensure the bottoms of the tubes are submerged. Timing is critical.
−
# Place 2ml tubes in float, and heat shock the cells by immersion in a pre-heated water bath at '''42ºC for 60 seconds'''.
+
# '''Incubate on ice for 5min:''' Return transformation tubes to ice bucket.
−
# Incubate the cells on ice for 5 minutes.
+
# '''Pipette 200µl SOC media to each transformation:''' SOC should be stored at 4°C, but can be warmed to room temperature before use. Check for contamination.  
−
# Add 200 μl of SOC media (''make sure that the SOC does not contain antibiotics and is not contaminated'') to each transformation, for a total of 250ul.
+
# '''Incubate at 37°C for 2 hours, shaker or rotor recommended:'''
−
# Incubate the cells at 37ºC for 2 hours while the tubes are rotating or shaking.  '''Important:''' ''2 hour recovery time helps in transformation efficiency, especially for plasmid backbones with antibiotic resistance other than ampicillin.''
+
# '''Pipette each transformation on two petri plates for a 20µl and 200µl plating:''' Pipette 20µl and 200µl of the transformation onto appropriately labeled plates. Spread with sterilized spreader or glass beads immediately. This helps ensure that you will be able to pick out a single colony.
−
# Label two petri dishes with LB agar and the appropriate antibiotic(s) with the part number, plasmid backbone, and antibiotic resistance.  Plate 20 µl and 200 µl of the transformation onto the dishes, and spread. This helps ensure that you will be able to pick out a single colony.
+
# '''Incubate transformations overnight (14-18hr) at 37°C:''' Incubate the plates upside down (agar side facing up). If incubated for too long, colonies may overgrow and the antibiotics may start to break down; un-transformed cells will begin to grow.  
−
# For the control, label two petri dishes with LB agar (with the appropriate antibiotic).  Plate 20 µl and 200 µl of the transformation onto the dishes, and spread.
+
# '''Pick single colonies:''' Pick single colonies from transformations: do a colony PCR to verify part size, make glycerol stocks, grow up cell cultures and miniprep.
−
# Incubate the plates at 37ºC for 12-14 hours, making sure the agar side of the plate is up. If incubated for too long the antibiotics start to break down and un-transformed cells will begin to grow. This is especially true for ampicillin - because the resistance enzyme is excreted by the bacteria, and inactivates the antibiotic outside of the bacteria.
+
# '''Count colonies for control transformation:''' Count colonies on the 20μl control plate and calculate your competent cell efficiency. Competent cells should have an efficiency of 1.5x10^8 to 6x10^8 cfu/µg DNA.
−
# You can pick single colonies, do a colony PCR, make a glycerol stock, grow up a cell culture and [[Help:Protocols/Miniprep|miniprep]].
+
−
# Count the colonies on the 20 μl '''control plate''' and calculate your competent cell efficiency.
+
  
  

Revision as of 20:31, 28 July 2015

Transformation Protocol

Printable PDF Protocol
Estimated bench time: 1 hour
Estimated total time: 3 hours (plus 14-18 hour incubation)

Transformations are essential to using the DNA Distribution Kits: resuspend the DNA sample in a well, transform the DNA into competent cells, and select single colonies. However, transformations can also be one of the more fickle laboratory techniques.


At iGEM HQ, we run test transformations of the DNA Distribution Kit with the following protocol. We have found that it is the best protocol to use with the DNA Distribution Kit and ensures high efficiency transformations.


  • At iGEM HQ, we make our own stocks of NEB 10b competent cells. Competent cells purchased from vendors will have better efficiency.
  • Make sure to test the competency of your cells with the provided Competent Cell Test Kit.
  • Read through the entire protocol before starting!


Materials

Material Description/Comments
Resuspended DNA Resuspend DNA Distribution Kit well(s) with 10µl dH20. Pipet up and down several times, let sit for a few minutes. Resuspension will be red from cresol red dye.
10pg/µl Control DNA 1µl for control transformation. pSB1C3 w/ BBa_J04450, RFP on high-copy chloramphenicol resistant plasmid. Located in the Competent Cell Test Kit.
Competent Cells 50µl per transformation. iGEM HQ stores competent cells in aliquots of 260µl (5rxns total) at -80°C.
2ml Microtubes One tube per transformation. Label tubes with part name or well location before starting.
Floating Foam Tube Rack Place 2ml tubes in floating tube rack for better support when working on ice and for the heat shock in the water bath.
Ice & ice bucket Fill bucket with ice, and pre-chill 2ml tubes (5min). Thaw competent cell stock on ice (10-15min).
Lab Timer
42°C water bath Set water bath to 42°C before starting.
SOC Media 200µl per transformation. SOC Media is better than LB Media for higher transformation efficiency. SOC Media should not contain antibiotics, and can be easily contaminated.
37°C incubator Preferably with a rotor/shaker for 2ml tubes. Incubate petri plates overnight (non-agitated).
Petri plates w/ LB agar and antibiotic 2 plates per transformation: for 20µl and 200µl platings. Make sure to use appropriate antibiotic. Label with part name or well location before starting.
Sterile spreader or glass beads Used to spread transformation across petri plates. Be sure to use sterile technique in between platings.
Pipettes and Tips 10µl, 20µl, 200µl tips and pipettes recommended


Setup & Protocol

When transforming competent cells, both timing and temperature are very important. Use a lab timer, follow the incubation temperatures closely, and keep materials on ice when required.


Resuspend DNA in selected wells in the Distribution Kit. Label 2ml tubes with part name or well location. Fill lab ice bucket with ice, and pre-chill 2ml tubes (one tube for each transformation, including your control) in a floating foam tube rack.


  1. Thaw competent cells on ice: This may take 10-15min for a 260µl stock. Dispose of unused competent cells. Do not refreeze unused thawed cells, as it will drastically reduce transformation efficiency.
  2. Pipette 50µl of competent cells into 2ml tube: 50µl in a 2ml tube per transformation. Tubes should be labeled, pre-chilled, and in a floating tube rack for support. Keep all tubes on ice. Don’t forget a 2ml tube for your control.
  3. Pipette 1µl of resuspended DNA into 2ml tube: Pipette from well into appropriately labeled tube. Gently pipette up and down a few times. Keep all tubes on ice.
  4. Pipette 1µl of control DNA into 2ml tube: Pipette 1µl of 10pg/µl control into your control transformation. Gently pipette up and down a few times. Keep all tubes on ice.
  5. Close 2ml tubes, incubate on ice for 30min: Tubes may be gently agitated/flicked to mix solution, but return to ice immediately.
  6. Heat shock tubes at 42°C for 1 min: 2ml tubes should be in a floating foam tube rack. Place in water bath to ensure the bottoms of the tubes are submerged. Timing is critical.
  7. Incubate on ice for 5min: Return transformation tubes to ice bucket.
  8. Pipette 200µl SOC media to each transformation: SOC should be stored at 4°C, but can be warmed to room temperature before use. Check for contamination.
  9. Incubate at 37°C for 2 hours, shaker or rotor recommended:
  10. Pipette each transformation on two petri plates for a 20µl and 200µl plating: Pipette 20µl and 200µl of the transformation onto appropriately labeled plates. Spread with sterilized spreader or glass beads immediately. This helps ensure that you will be able to pick out a single colony.
  11. Incubate transformations overnight (14-18hr) at 37°C: Incubate the plates upside down (agar side facing up). If incubated for too long, colonies may overgrow and the antibiotics may start to break down; un-transformed cells will begin to grow.
  12. Pick single colonies: Pick single colonies from transformations: do a colony PCR to verify part size, make glycerol stocks, grow up cell cultures and miniprep.
  13. Count colonies for control transformation: Count colonies on the 20μl control plate and calculate your competent cell efficiency. Competent cells should have an efficiency of 1.5x10^8 to 6x10^8 cfu/µg DNA.


Other Resources

Video

Transforming Your Part from iGEM Videos.

  • Please note, this video may be outdated.