IGEM:Paris Bettencourt 2012/Notebooks/MAGE group: Difference between revisions
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=25/06/12= | =25/06/12= | ||
looking for MAGE protocols | looking for MAGE protocols | ||
from past iGEM team | ==from past iGEM team == | ||
==Harvard 2011 | ===Harvard 2011 === | ||
[http://2011.igem.org/Team:Harvard/Protocols#MAGE] | [http://2011.igem.org/Team:Harvard/Protocols#MAGE] | ||
===MAGE=== | ====MAGE==== | ||
====MAGE oligo strand choice==== | =====MAGE oligo strand choice===== | ||
'''Choosing the strand''' | '''Choosing the strand''' | ||
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*For HisB deletion, we used the same strand, and for the rpoZ deletion, we used the reverse complement. | *For HisB deletion, we used the same strand, and for the rpoZ deletion, we used the reverse complement. | ||
====Protocol==== | =====Protocol===== | ||
# Grow up cells to mid-log at 30C. | # Grow up cells to mid-log at 30C. | ||
# Induce lambda red machinery by incubating cells at 42C for 15 minutes (for other strains, induction procedure may be different). | # Induce lambda red machinery by incubating cells at 42C for 15 minutes (for other strains, induction procedure may be different). | ||
| Line 26: | Line 26: | ||
==Yale 2011 | ===Yale 2011 === | ||
*"Received 22 degenerate oligonucleotide sequences from Keck and performed MAGE. Cultures were grown up to midlog post electroporation and freeze-thawed approximately 14 times before plating. Suriving colonies were isolated, and we will soon put them through another cycle of MAGE. The process will be repeated. " | *"Received 22 degenerate oligonucleotide sequences from Keck and performed MAGE. Cultures were grown up to midlog post electroporation and freeze-thawed approximately 14 times before plating. Suriving colonies were isolated, and we will soon put them through another cycle of MAGE. The process will be repeated. " | ||
Revision as of 06:52, 25 June 2012
25/06/12
looking for MAGE protocols
from past iGEM team
Harvard 2011
MAGE
MAGE oligo strand choice
Choosing the strand
MAGE is much more efficient if the lagging strand is targeted with the MAGE oligo. It is thought that the MAGE oligo takes the place of the RNA primer that begins Okazaki fragments on the lagging strand during DNA replication. The diagram below shows which strand to choose when designing MAGE oligos. Use ecocyc.org[2] to determine where on the genome your site of interest lies, and which strand it's on (determined by forward or reverse orientation in the genome browser). Examples
- For HisB deletion, we used the same strand, and for the rpoZ deletion, we used the reverse complement.
Protocol
- Grow up cells to mid-log at 30C.
- Induce lambda red machinery by incubating cells at 42C for 15 minutes (for other strains, induction procedure may be different).
- Centrifuge 1-1.5mL of culture at 4C for 1 minute at top speed
- Remove all the media from the pellet and resuspend in 1mL of cold ddH2O before spinning again at 4C for 1 min at top speed.
- Repeat for a second water wash.
- Remove water carefully, taking care not to disturb the cell pellet. Add the MAGE oligo (amount will vary: add enough to have a final concentration of 2.5μM in a total volume of 50μL) and cold ddH2O to bring the volume up to 50μL. Mix and transfer to a cold, 1mm gap cuvette for electroporation.
-Note: MAGE oligo should preferentially be concentrated enough that only a few microliters need to be added. Adding larger amounts, especially if the salt concentration is high, may interfere with electroporation.
- Thoroughly dry the sides of the cuvette and electroporate at 1.80 kV for ideally 5.7 ms (Ec1 setting on many electroporators).
- Immediately add 1mL of plain LB to the cuvette, pipette up and down to mix, and transfer to a culture tube containing an additional 2mL of plain LB.
- Recover at 30C at least 1 hour before plating, or if performing additional rounds of MAGE, let culture grow until it reaches mid-log and repeat procedure.
Yale 2011
- "Received 22 degenerate oligonucleotide sequences from Keck and performed MAGE. Cultures were grown up to midlog post electroporation and freeze-thawed approximately 14 times before plating. Suriving colonies were isolated, and we will soon put them through another cycle of MAGE. The process will be repeated. "
