Difference between revisions of "Transformation"

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*      Which traits do we want in the transformants, especially with regards to having an intact lacZ gene?  
 
*      Which traits do we want in the transformants, especially with regards to having an intact lacZ gene?  
 
*      Which cells will these be put into? (traditionally, DH5alpha for plasmid work and BL21 pLysS for protein expression)  
 
*      Which cells will these be put into? (traditionally, DH5alpha for plasmid work and BL21 pLysS for protein expression)  
 +
*      Do we have a dilution of the positive control DNA? If not, use the DNA strain database to find the concentration of DNA in the stock of your plasmid of interest and use that to calculate a dilution to give you 25ngDNA/10µL  (equal to 2.5ng DNA/µL).
  
 
Current version (Version 2):
 
Current version (Version 2):
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# After 30 mins, carry the ice beaker to a heat block pre-heated to 42ºC. and heat shock the cells for exactly 45 seconds, then immediately return the tubes to ice for 2 minutes.  
 
# After 30 mins, carry the ice beaker to a heat block pre-heated to 42ºC. and heat shock the cells for exactly 45 seconds, then immediately return the tubes to ice for 2 minutes.  
 
# Add 250 ul of room temperature LB to each of your tubes.
 
# Add 250 ul of room temperature LB to each of your tubes.
# Incubate the tubes in the large floor shaking incubator set to 37°C, on their side. Make sure tubes are completely shut.  
+
# Incubate the tubes in the large floor shaking incubator set to 34°C, on their side. Make sure tubes are completely shut.  
# After 60 minutes, plate cells. Divide ligated plasmids that are going into DH5alpha cells into three plates of 10µL, 100µL, and the rest of the volume. The entire volume can be plated onto one plate for Gibbon Assembly plasmids and for BL21 cells.  
+
# After 60 minutes, plate cells. Divide ligated plasmids that are going into DH5alpha cells into three plates of 10µL, 100µL, and the rest of the volume (260µL). The entire volume can be plated onto one plate for Gibbon Assembly plasmids and for BL21 cells.  
# Spread plates with sterile beads, and then pour used beads into white container with funnel (by the sink).
+
# Spread plates with sterile beads. Put lid on plate and flip over so beads fall onto lid. Pour used beads into white container with funnel (by the sink).
# Incubate overnight. Gibson Assembly may take an extra night to see colonies.  
+
# Incubate overnight. You can use the 37°C incubator with ambient CO2, located near the weigh station (only if you are transforming a nonpathogenic bacteria). Gibson Assembly may take an extra night to see colonies.  
 
''Note: If you are using Kanamycin plates, a large number small colonies will appear after 48 to 72 hours. These are not transformed colonies. We are not sure why this happens. Feel free to figure it out.''
 
''Note: If you are using Kanamycin plates, a large number small colonies will appear after 48 to 72 hours. These are not transformed colonies. We are not sure why this happens. Feel free to figure it out.''
  

Latest revision as of 03:19, 8 November 2019

Transformation

First, answer these questions:

  • Which type of LB plate is required?
  • Which traits do we want in the transformants, especially with regards to having an intact lacZ gene?
  • Which cells will these be put into? (traditionally, DH5alpha for plasmid work and BL21 pLysS for protein expression)
  • Do we have a dilution of the positive control DNA? If not, use the DNA strain database to find the concentration of DNA in the stock of your plasmid of interest and use that to calculate a dilution to give you 25ngDNA/10µL (equal to 2.5ng DNA/µL).

Current version (Version 2):

  1. Create enough LB plates with the appropriate antibiotic, as well as IPTG and X-gal if you are using blue/white screening. If you are doing a low number of transformants, these chemicals can be added directly to normal LB plates, with beads to spread out the chemicals. Wait at least one hour for the chemicals (antibiotics and possibly IPTG + X-gal) to diffuse into the plates.
  2. Turn on heat block to 42ºC.
  3. Turn on the large shaking incubator to 37ºC.
  4. Spray a beaker with ethanol, go get ice from the second floor imaging room (across from lab).
  5. From the -20 freezer: get one tube of competent cells for each transformation, plus one for a positive control (intact plasmid only), plus one for a negative control (water only). Each freezer tube has 100uL cells. Place tubes into the ice.
  6. Add 10ul of ligated plasmid (should be ~25ng) DNA to one tube of cells, and repeat for each transformation. Also add 25ng of empty vector to a separate tube of cells. Add 10uL of sterile water to the negative control tubes. Place back on ice. As you add these small volumes, mix gently with tip of your pipette.
  7. Allow the cells to incubate for 30 minutes on ice. Do not mix the tubes during this incubation period.
  8. After 30 mins, carry the ice beaker to a heat block pre-heated to 42ºC. and heat shock the cells for exactly 45 seconds, then immediately return the tubes to ice for 2 minutes.
  9. Add 250 ul of room temperature LB to each of your tubes.
  10. Incubate the tubes in the large floor shaking incubator set to 34°C, on their side. Make sure tubes are completely shut.
  11. After 60 minutes, plate cells. Divide ligated plasmids that are going into DH5alpha cells into three plates of 10µL, 100µL, and the rest of the volume (260µL). The entire volume can be plated onto one plate for Gibbon Assembly plasmids and for BL21 cells.
  12. Spread plates with sterile beads. Put lid on plate and flip over so beads fall onto lid. Pour used beads into white container with funnel (by the sink).
  13. Incubate overnight. You can use the 37°C incubator with ambient CO2, located near the weigh station (only if you are transforming a nonpathogenic bacteria). Gibson Assembly may take an extra night to see colonies.

Note: If you are using Kanamycin plates, a large number small colonies will appear after 48 to 72 hours. These are not transformed colonies. We are not sure why this happens. Feel free to figure it out.



Older version 1:

  1. Create enough LB plates with the appropriate antibiotic, as well as IPTG and X-gal if you are using blue/white screening. If you are doing a low number of transformants, these chemicals can be added directly to normal LB plates, with beads to spread out the chemicals. Wait at least one hour for the chemicals (antibiotics and possibly IPTG + X-gal) to diffuse into the plates.
  2. Turn on heat block to 42ºC.
  3. Turn on shaking incubator to 37ºC.
  4. Turn on and close lid of refrigerated centrifuge, to allow it to start chilling down to 4ºC.
  5. Spray a beaker with ethanol, go get ice from the second floor imaging room (across from lab).
  6. Label 1 1.5mL microfuge tube for each of your experimental groups. (3 tubes for pUC18, pUC18-Empty, water 1 Control). From the -80 freezer: get enough competent cells to transfer 250 uL cells in each of the previously labeled experimental group tubes. Each freezer tube has 100uL cells. For negative control (water) you can use 100uL cells.
  7. Transfer 250uL of bacterial cells to each of your ligated and empty plasmid microfuge tubes. Transfer 100uL to the water tube. Spin for 5 minutes at 10,000 rpm at 4ºC. This will cause the bacterial cells to collect in a pellet at the bottom of your tube.
  8. Remove the supernatant without disrupting the bacterial pellets. Add 25 µL of ice cold 1x TSS buffer/tube (10uL for the water tube) and resuspend the cells by flicking the tube gently, then place the tubes on ice. [TSS buffer is LB with detergents and salts which will permeabilize cell membranes, allowing the plasmid to enter the bacterial cells].
  9. Add 10µL (25ng) of ligated plasmid DNA to the cells in ligated plasmid group. Also add 25ng of empty vector to the cells in the empty vector tubes. Add 10uL of sterile water to the negative control tubes. Place back on ice. As you add these small volumes, mix gently with tip of your pipette.
  10. Allow the cells to incubate for 40 minutes on ice. Do not mix the tubes during this incubation period.
  11. After 40 mins, carry the ice bucket to a heat block pre-heated to 42ºC. and heat shock the cells for exactly 45 seconds, then immediately return the tubes to ice for 2 minutes. Add 250 ul of room temperature LB to each of your tubes.
  12. Incubate your microcentrifuge tubes with cells in the large floor shaking incubator set to 37°C, on their side. Make sure tubes are completely shut.
  13. After 60 minutes, plate cells. If you so choose, you can divide the volume of ~250µL LB/cells/DNA mixture into a plate of 10µL, 100µL, and the rest of the volume in order to see which volume grows the optimal amount of cells. Spread plates with sterile beads, and then pour used beads into white container with funnel (by the sink).