Protocols


Procedure (pdf version)
ProcedureRecombineering
Posted On12/28/2010 3:09 PM
AuthorLei Sun
Science WriterNora G. Smart
Background

Recombineering (recombination-mediated genetic engineering) is a powerful method for fast and efficient construction of vectors for subsequent manipulation of the mouse genome or for use in cell culture experiments. This protocol is modified from the posted ones on http://web.ncifcrf.gov/research/brb/recombineeringInformation.aspx. More details about this technology are also available on this site.

Reagents and Solutions

LB Broth and LB Agar:

Sigma

 

Sucrose media (liquid):

1% Trypton

0.5% Yeast Extraction

6% Sucrose

 

Sucrose media (Solid):

1% Trypton

0.5% Yeast Extraction

6% Sucrose

1% Agar

Method

Transformation of BAC into Recombinogenic Strain SW102

  1. Make a 5 ml o/n culture either from the SW102 freeze stock or from a single colony.
  2. Dilute the o/n culture 1:50. Incubate with appropriate antibiotic selection in at 32°C shaking water bath until the density reaches an OD600 of 0.6.
  3. Cooled down the SW102 culture in the ice/water bath slurry and subsequently transferred into pre-cooled 14 ml Falcon Round-Bottom tubes.
  4. Spin down the bacteria at 0°C, 4000 RPM for 5 min.
  5. Pour off the supernatant and briefly invert tube on a paper towel, and add 1 ml ice-cold ddH2O. Leave the tube on ice for 3 min and resuspend the pellet in the ddH2O by gently swirling the tube in the ice/water bath. Add another 9 ml ice-cold ddH2O and pellet the samples again.
  6. Pour off the supernatant, resuspend as in step 5 and spin twice more.
  7. Remove all supernatant by inverting the tube on a paper towel. Gently resuspend the pellet as before to a final volume of 50 μl.
  8. Mix 25 μl of the freshly made electrocompetent cells with 100ng BAC DNA. Transfer the cocktail to a pre-cooled 0.1 cm cuvette.
  9. Transform by electroporation at 25 F, 1.75 kV, and 200 ohms, twice.
  10. Transfer the bacteria to a tube with 1 ml LB medium. Incubate at 32°C for 1 h.
  11. Plate serial dilutions (100 μl, 100 μl of a 1:10 dilution, 100 μl 1:100, and 100 μl 1:1000) the transformed bacteria on chloramphenicol (12.5 μg/ml) LB medium.
  12. Incubate at 32°C for 36-48 hours

 

Insertion of Kan-SacB Cassette

  1. PCR amplify the Kan-SacB cassette with Phusion High Fidelity. Use 1-2 ng templates (the pDCE069 plasmid). 98°C 30 sec., (98°C 30 sec., 57°C 30 sec., 72°C 3.5 min.) for 40 cycles, 72°C 10 min. Gel-purify the PCR product, and elute in 30 μl ddH2O.
  2. Inoculate an overnight culture of SW102 cells containing the BAC. 5 ml LB + chloramphenicol. Incubate at 32°C.
  3. Next day, turn on two shakers: One at 32°C, the other at 42°C. Dilute 500 μl of the overnight culture in 25 ml LB with chloramphenicol (12.5 μg/ml), and incubate at 32°C to an OD600 of 0.6.
  4. Transfer 10 ml to another flask and heat-shock at 42°C for exactly 15 min. in a shaking water bath. The remaining culture is left at 32°C as the un-induced control.
  5. After 15 min., the two samples, induced and un-induced, are briefly cooled in an ice/waterbath slurry and then transferred to two 14 ml Falcon Round-Bottom tubes and pelleted using 4000 RPM at 0°C for 5 min.
  6. Pour off the supernatant and resuspend the pellet in 1 ml ice-cold ddH2O by gently swirling the tubes in the ice/waterbath slurry. No pipetting. When resuspended, add another 9 ml ice-cold ddH2O and pellet the samples again.
  7. Repeat step 10 twice.
  8. Remove all supernatant by inverting the tube on a paper towel. Gently resuspend the pellet as before to a final volume of 50 μl.
  9. Mix 25 μl of the freshly made electrocompetent cells with 200~300ng Kan-SacB fragment. Transfer the cocktail to a pre-cooled 0.1 cm cuvette.
  10. Transform by electroporation at 25 μF, 1.75 kV, and 200 ohms, twice.
  11. Transfer the bacteria to a tube with 1 ml LB medium. Incubate at 32°C for 1 h.
  12. Plate serial dilutions (100 μl, 100 μl of a 1:10 dilution, 100 μl 1:100) the transformed bacteria on chloramphenicol (12.5 μg/ml) and kanamycin (25 μg/ml) LB medium.
  13. Incubate at 32°C for 36-48 hours.

 

Replacement of Kan-SacB Cassette

  1. Inoculate an overnight culture of SW102 cells containing the BAC with Kan-SacB fragment. 5 ml LB + chloramphenicol. Incubate at 32°C.
  2. Next day, turn on two shakers: One at 32°C, the other at 42°C. Dilute 500 ?l of the overnight culture in 25 ml LB with chloramphenicol (12.5 μg/ml) and kanamycin (25 μg/ml), and incubate at 32°C to an OD600 of 0.6.
  3. Transfer 10 ml to another flask and heat-shock at 42°C for exactly 15 min. in a shaking water bath.
  4. After 15 min., the two samples, induced and un-induced, are briefly cooled in an ice/waterbath slurry and then transferred to two 14 ml Falcon Round-Bottom tubes and pelleted using 4000 RPM at 0°C for 5 min.
  5. Pour off the supernatant and resuspend the pellet in 1 ml ice-cold ddH2O by gently swirling the tubes in the ice/waterbath slurry. No pipetting. When resuspended, add another 9 ml ice-cold ddH2O and pellet the samples again.
  6. Repeat step 10 twice.
  7. Remove all supernatant by inverting the tube on a paper towel. Gently resuspend the pellet as before to a final volume of 50 μl.
  8. Mix 25 μl of the freshly made electrocompetent cells with 200~300ng DNA fragment (PCR product for marker insertion, and oligo for point mutation). Transfer the cocktail to a pre-cooled 0.1 cm cuvette.
  9. Transform by electroporation at 25 μF, 1.75 kV, and 200 ohms, twice.
  10. Transfer the bacteria to a tube with 1 ml Sucrose medium. Incubate at 32°C for 1 h.
  11. Plate serial dilutions (100 μl, 100 μl of a 1:10 dilution, 100 μl 1:100, and 100 μl 1:1000) the transformed bacteria on chloramphenicol (12.5 μg/ml) Sucrose medium.
  12. Incubate at 32°C for 36-48 hours.
Critical Parameters and Troubleshooting

Keep the cells at 32°C or lower.