Difference between revisions of "Help:Protocols/Transformation"

m
 
(18 intermediate revisions by 3 users not shown)
Line 1: Line 1:
−
__NOTOC__
+
[[Category:Protocols]]
 +
{{TOCright}}
 
{{HelpPage/MainLinks}}
 
{{HelpPage/MainLinks}}
  
−
=Transformation Protocol=
+
<html>
−
'''Estimated time: 3 hours''' (plus 14-18 hour incubation)<br>
+
<style type="text/css">
 +
.protocolTable {
 +
border: 1px solid #ccc;
 +
border-collapse: collapse;
 +
width: auto;
 +
margin: 15px 10px;;
 +
}
  
−
Transformations are essential to using the [[Help:Distribution_Kits|distribution kits]] sent out by the Registry. They can also be one of the more fickle laboratory techniques. We recommend the following protocol as it is the same one used at iGEM HQ.
+
.protocolTable  td {
 +
padding: 5px;
 +
border: 1px solid #ccc;
 +
border-collapse: collapse;
 +
vertical-align: text-top;
 +
}
  
−
We have tested transformations of the distribution kit with this protocol and have found that it is the best protocol to use with Registry parts and ensures the highest efficiency for the transformation. This protocol may be particularly useful if you are finding that your transformations are not working, or yielding few colonies.
+
.protocolTable th {
 +
background-color: #464646;
 +
padding: 5px;
 +
color: #f2f2f2;
 +
border: 1px solid #ccc;
 +
border-collapse: collapse;
 +
vertical-align: text-top;
 +
text-align: left;
 +
}
 +
.protocolTable  td {
 +
padding: 5px;
 +
border: 1px solid #ccc;
 +
border-collapse: collapse;
 +
vertical-align: text-top;
 +
}
 +
.protocolTable  .material {
 +
font-weight: bold;
 +
}
 +
</style>
 +
</html>
 +
=Overview=
 +
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 make our own stocks of NEB 10beta competent cells. Competent cells purchased from vendors will have better efficiency.
 
  
−
<html>
+
At iGEM HQ, we have run test transformations of the DNA Distribution Kit with the following protocols. We have found that these are the best protocols to use with the DNA Distribution Kit to ensure high efficiency transformations. We provide two alternative protocols: Single-tube reactions, for transforming a few samples at a time; and 96-well format transformations, for transforming multiple samples at once. The 96-well format transformation protocol is also appropriate for use with single-tube reactions in labs where there is no access to a 37°C shaker-incubator or microcentrifuge.
−
<iframe src="http://player.vimeo.com/video/25201947" width="320" height="180" frameborder="0" webkitAllowFullScreen mozallowfullscreen allowFullScreen></iframe> <p><a href="https://igem.org/Videos/Transforming_Your_Part">Transforming Your Part</a> from <a href="https://igem.org/Videos">iGEM Videos</a>.</p>
+
 
−
</html>
+
 
 +
*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]].
 +
*'''Read through the entire protocol before starting!'''
 +
 
 +
If you have issues with transformations, read through our [[Help:Protocols/Transformation/Troubleshooting|Troubleshooting]] page for help.
 +
 
 +
=Single Tube Transformation Protocol=
 +
 
 +
==Before You Start==
 +
 
 +
'''Estimated bench time: 1 hour'''<br />
 +
'''Estimated total time: 2 hours''' (plus 14-18 hour incubation)<br />
 +
 
 +
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.  
 +
*'''Read through the entire protocol before starting!'''
  
 
==Materials==
 
==Materials==
−
*Resuspended DNA (''Resuspend well in 10ul dH20, pipette up and down several times, let sit for a few minutes'')
+
*Resuspended DNA to be transformed
−
*[[Help:Protocols/Competent_Cells | Competent cells]] (''50ul per transformation'')
+
*10pg/µl Positive transformation control DNA (e.g. pSB1C3 w/ BBa_J04450, RFP on high-copy chloramphenicol resistant plasmid. Located in the [[Help:Competent_Cell_Test_Kit|Competent Cell Test Kit]].)
−
*Ice (in ice bucket/container)
+
*Competent Cells (50µl per sample)
−
*2ml tube (''1 per a transformation''')
+
*1.5mL Microtubes
−
*42ºC water bath
+
*SOC Media (950µL per sample)
−
*SOC media (''check for contamination!'')
+
*Petri plates w/ LB agar and antibiotic (2 per sample)
−
*Petri dishes with LB agar and appropriate antibiotic (''2 per transformation'')
+
−
*glass beads or spreader
+
−
*37ºC incubator
+
−
*10pg/ul RFP Control (pSB1A3 w/ BBa_J04450)
+
  
−
==Procedure==
+
==Equipment==
−
# Start thawing the competent cells on ice.  
+
*Floating Foam Tube Rack
−
# Add 50 µL of thawed competent cells into pre-chilled 2ml tube, and another 50µL into a 2ml tube, labelled for your control.
+
*Ice & ice bucket
−
# Add 1 - 2 µL of the resuspended DNA to the 2ml tube. Pipet up and down a few times, gently. Make sure to keep the competent cells on ice.
+
*Lab Timer
−
# Add 1 µL of the RFP Control to your control transformation.  
+
*42°C water bath
−
# Close tubes and incubate the cells on ice for 30 minutes.
+
*37°C incubator
−
# Heat shock the cells by immersion in a pre-heated water bath at 42ºC for 60 seconds.
+
*Sterile spreader or glass beads
−
# Incubate the cells on ice for 5 minutes.
+
*Pipettes and Tips (10µl, 20µl, 200µl recommended)
−
# Add 200 μl of SOC media (make sure that the broth does not contain antibiotics and is not contaminated) to each transformation
+
*Microcentrifuge
−
# 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.''
+
 
−
# 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.
+
==Method==
−
# For the control, label two petri dishes with LB agar (AMP).  Plate 20 µl and 200 µl of the transformation onto the dishes, and spread.
+
 
−
# 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.
+
#Resuspend DNA in selected wells in the Distribution Kit with 10µl dH20. Pipet up and down several times, let sit for a few minutes. Resuspension will be red from cresol red dye.
−
# You can pick a single colony, make a glycerol stock, grow up a cell culture and [[Help:Protocols/Miniprep|miniprep]].
+
#Label 1.5ml tubes with part name or well location. Fill lab ice bucket with ice, and pre-chill 1.5ml tubes (one tube for each transformation, including your control) in a floating foam tube rack.
−
# Count the colonies on the 20 μl control plate and calculate your competent cell efficiency.
+
# '''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.
 +
# '''Pipette 50µl of competent cells into 1.5ml tube:''' 50µl in a 1.5ml 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 1.5ml tube for your control.
 +
# '''Pipette 1µl of resuspended DNA into 1.5ml tube:''' Pipette from well into appropriately labeled tube. Gently pipette up and down a few times. Keep all tubes 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.
 +
# '''Close 1.5ml tubes, incubate on ice for 30min:''' Tubes may be gently agitated/flicked to mix solution, but return to ice immediately.
 +
# '''Heat shock tubes at 42°C for 45 sec:''' 1.5ml 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.
 +
# '''Incubate on ice for 5min:''' Return transformation tubes to ice bucket.
 +
# '''Pipette 950µl SOC media to each transformation:''' SOC should be stored at 4°C, but can be warmed to room temperature before use. Check for contamination.
 +
# '''Incubate at 37°C for 1 hours, shaking at 200-300rpm'''
 +
# '''Pipette 100µL of each transformation onto petri plates''' Spread with sterilized spreader or glass beads immediately. This helps ensure that you will be able to pick out a single colony.
 +
# '''Spin down cells at 6800g for 3mins and discard 800µL of the supernatant. Resuspend the cells in the remaining 100µL, and pipette each transformation onto petri plates''' Spread with sterilized spreader or glass beads immediately. This increases the chance of getting colonies from lower concentration DNA samples.
 +
# '''Incubate transformations overnight (14-18hr) at 37°C:''' Incubate the plates upside down (agar side up). If incubated for too long, colonies may overgrow and the antibiotics may start to break down; un-transformed cells will begin to grow.
 +
# '''Pick single colonies:''' Pick single colonies from transformations: do a colony PCR to verify part size, make glycerol stocks, grow up cell cultures and [[Help:Protocols/Miniprep|miniprep]].
 +
# '''Count colonies for control transformation:''' Count colonies on the 100μl control plate and [[Help:Competent_Cell_Test_Kit|calculate your competent cell efficiency]]. Competent cells should have an efficiency of 1.5x10^8 to 6x10^8 cfu/µg DNA.
 +
 
 +
 
 +
=96-Well Transformation Protocol=
 +
 
 +
==Before You Start==
 +
 
 +
[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 />
 +
 
 +
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.
 +
*'''Read through the entire protocol before starting!'''
 +
 
 +
==Materials==
 +
*Resuspended DNA to be transformed
 +
*10pg/µl Positive transformation control DNA
 +
*Competent Cells (50µl per sample)
 +
*Flat-bottomed 200µL 96-well plate OR 2ml Microtubes
 +
*SOC Media (200µL per sample)
 +
*Petri plates w/ LB agar and antibiotic (2 per sample)
 +
 
 +
==Equipment==
 +
*Floating Foam Tube Rack
 +
*Ice & ice bucket
 +
*Lab Timer
 +
*42°C water bath
 +
*37°C incubator
 +
*Sterile spreader or glass beads
 +
*Pipettes and Tips (10µl, 20µl, 200µl tips and pipettes recommended)
 +
 
 +
==Method==
 +
 
 +
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.
 +
 
 +
 
 +
# '''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.
 +
# '''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.
 +
# '''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.
 +
# '''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.
 +
# '''Close 2ml tubes, incubate on ice for 30min:''' Tubes may be gently agitated/flicked to mix solution, but return to ice immediately.
 +
# '''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.
 +
# '''Incubate on ice for 5min:''' Return transformation tubes to ice bucket.
 +
# '''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.
 +
# '''Incubate at 37°C for 2 hours, shaker or rotor recommended:'''
 +
# '''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.
 +
# '''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.  
 +
# '''Pick single colonies:''' Pick single colonies from transformations: do a colony PCR to verify part size, make glycerol stocks, grow up cell cultures and [[Help:Protocols/Miniprep|miniprep]].
 +
# '''Count colonies for control transformation:''' Count colonies on the 20μl control plate and [[Help:Competent_Cell_Test_Kit|calculate your competent cell efficiency]]. Competent cells should have an efficiency of 1.5x10^8 to 6x10^8 cfu/µg DNA.
 +
 
 +
 
 +
 
 +
=Other Resources=
 +
 
 +
==Video==
 +
<html>
 +
<iframe src="http://player.vimeo.com/video/25201947" width="320" height="180" frameborder="0" webkitAllowFullScreen mozallowfullscreen allowFullScreen></iframe> <p><a href="https://igem.org/Videos/Transforming_Your_Part">Transforming Your Part</a> from <a href="https://igem.org/Videos">iGEM Videos</a>.</p>
 +
</html>
 +
*''Please note, this video may be outdated.''

Latest revision as of 18:14, 28 June 2017

Overview

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 have run test transformations of the DNA Distribution Kit with the following protocols. We have found that these are the best protocols to use with the DNA Distribution Kit to ensure high efficiency transformations. We provide two alternative protocols: Single-tube reactions, for transforming a few samples at a time; and 96-well format transformations, for transforming multiple samples at once. The 96-well format transformation protocol is also appropriate for use with single-tube reactions in labs where there is no access to a 37°C shaker-incubator or microcentrifuge.


  • 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!

If you have issues with transformations, read through our Troubleshooting page for help.

Single Tube Transformation Protocol

Before You Start

Estimated bench time: 1 hour
Estimated total time: 2 hours (plus 14-18 hour incubation)

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.

  • Read through the entire protocol before starting!

Materials

  • Resuspended DNA to be transformed
  • 10pg/µl Positive transformation control DNA (e.g. pSB1C3 w/ BBa_J04450, RFP on high-copy chloramphenicol resistant plasmid. Located in the Competent Cell Test Kit.)
  • Competent Cells (50µl per sample)
  • 1.5mL Microtubes
  • SOC Media (950µL per sample)
  • Petri plates w/ LB agar and antibiotic (2 per sample)

Equipment

  • Floating Foam Tube Rack
  • Ice & ice bucket
  • Lab Timer
  • 42°C water bath
  • 37°C incubator
  • Sterile spreader or glass beads
  • Pipettes and Tips (10µl, 20µl, 200µl recommended)
  • Microcentrifuge

Method

  1. Resuspend DNA in selected wells in the Distribution Kit with 10µl dH20. Pipet up and down several times, let sit for a few minutes. Resuspension will be red from cresol red dye.
  2. Label 1.5ml tubes with part name or well location. Fill lab ice bucket with ice, and pre-chill 1.5ml tubes (one tube for each transformation, including your control) in a floating foam tube rack.
  3. 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.
  4. Pipette 50µl of competent cells into 1.5ml tube: 50µl in a 1.5ml 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 1.5ml tube for your control.
  5. Pipette 1µl of resuspended DNA into 1.5ml tube: Pipette from well into appropriately labeled tube. Gently pipette up and down a few times. Keep all tubes on ice.
  6. 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.
  7. Close 1.5ml tubes, incubate on ice for 30min: Tubes may be gently agitated/flicked to mix solution, but return to ice immediately.
  8. Heat shock tubes at 42°C for 45 sec: 1.5ml 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.
  9. Incubate on ice for 5min: Return transformation tubes to ice bucket.
  10. Pipette 950µl SOC media to each transformation: SOC should be stored at 4°C, but can be warmed to room temperature before use. Check for contamination.
  11. Incubate at 37°C for 1 hours, shaking at 200-300rpm
  12. Pipette 100µL of each transformation onto petri plates Spread with sterilized spreader or glass beads immediately. This helps ensure that you will be able to pick out a single colony.
  13. Spin down cells at 6800g for 3mins and discard 800µL of the supernatant. Resuspend the cells in the remaining 100µL, and pipette each transformation onto petri plates Spread with sterilized spreader or glass beads immediately. This increases the chance of getting colonies from lower concentration DNA samples.
  14. Incubate transformations overnight (14-18hr) at 37°C: Incubate the plates upside down (agar side up). If incubated for too long, colonies may overgrow and the antibiotics may start to break down; un-transformed cells will begin to grow.
  15. 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.
  16. Count colonies for control transformation: Count colonies on the 100μl control plate and calculate your competent cell efficiency. Competent cells should have an efficiency of 1.5x10^8 to 6x10^8 cfu/µg DNA.


96-Well Transformation Protocol

Before You Start

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

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.

  • Read through the entire protocol before starting!

Materials

  • Resuspended DNA to be transformed
  • 10pg/µl Positive transformation control DNA
  • Competent Cells (50µl per sample)
  • Flat-bottomed 200µL 96-well plate OR 2ml Microtubes
  • SOC Media (200µL per sample)
  • Petri plates w/ LB agar and antibiotic (2 per sample)

Equipment

  • Floating Foam Tube Rack
  • Ice & ice bucket
  • Lab Timer
  • 42°C water bath
  • 37°C incubator
  • Sterile spreader or glass beads
  • Pipettes and Tips (10µl, 20µl, 200µl tips and pipettes recommended)

Method

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.