Smolke:Protocols/Library transformation

From OpenWetWare

< Smolke:Protocols(Difference between revisions)
Jump to: navigation, search
Current revision (17:05, 20 October 2009) (view source)
 
(One intermediate revision not shown.)
Line 22: Line 22:
#Clean up PCR product
#Clean up PCR product
#*Commercial kits work just fine here
#*Commercial kits work just fine here
-
#Using PCR product as template, set up 8x 100uL PCRs using Taq as the polymerase.
+
#Using PCR product as template, set up 8x 100uL PCRs using Pfu as the polymerase.
 +
#*For a 2.5kb template, reamplification with Taq introduced roughly 0.6 nucleotide mutations per template. Switching to Pfu made this step effectively faithful.
#Combine all 800uL of PCR product (generally 40-50μg of DNA) into one tube.
#Combine all 800uL of PCR product (generally 40-50μg of DNA) into one tube.

Current revision

Home        Contact        Internal        Protocols        Lab Members        Publications        Research       


Contents

Overview

Constructing large mutant libraries in S. cerevisiae by gap repair.

Materials

  • Mutazyme (Stratagene 200550)
  • 2mm gap electroporation cuvettes
  • Tris-DTT
    • 2.5M DTT, 1M Tris, pH 8.0. Dissolve 0.39g DTT in 1M Tris (pH 8.0) to a final volume of 1mL (usually ~600uL Tris). Filter sterilize, can store at -20°C for a few months.
  • Buffer E
    • 10mM Tris (pH 7.5), 2mM MgCl2, 270mM sucrose. Combine 46.2g sucrose, 5mL 1M Tris (pH 7.5), and 1mL 1M MgCl2, adjust volume to 500mL, and filter sterilize. Store at RT.

Procedure

DNA Prep

Best done the day before you plan to do your transformations. The backbone digest should run overnight (finishing the morning of the transformations). You can run the second round of PCRs on the day of the transformation, but I like to do them overnight as well (so they're ready the morning of).

Insert

  1. Follow directions on Mutazyme kit
    • Ideal mutation rate will depend on target and screening strategy
  2. DpnI treat the PCR product
    • Mutazyme can require large amounts of template. Adjust DpnI time accordingly
  3. Clean up PCR product
    • Commercial kits work just fine here
  4. Using PCR product as template, set up 8x 100uL PCRs using Pfu as the polymerase.
    • For a 2.5kb template, reamplification with Taq introduced roughly 0.6 nucleotide mutations per template. Switching to Pfu made this step effectively faithful.
  5. Combine all 800uL of PCR product (generally 40-50μg of DNA) into one tube.

Backbone

  1. Cut 8μg of vector backbone overnight.
    • Generally 400uL digest. Try to cut just inside the PCR primer binding sites. To be safe, I often add a third enzyme to cleave the middle of the insert.

Coprecipitation

  1. Run a gel to check that your PCR and digest worked
  2. Assuming success, make three tubes of DNA:
    1. 375uL insert + 150uL vector
    2. 375uL insert + 150uL vector
    3. 450uL water + 75uL vector (Negative control)
  3. Phenol-chloroform extract and ethanol precipitate tubes (can't use commercial columns since it's so much DNA)
    • DO NOT resuspend - you'll use the cell suspension to resuspend the DNA later. Just leave the DNA precipitated in the tube

Transformation

  1. Set up an overnight culture of your yeast strain (5mL is fine)
  2. In the morning, dilute back to 50mL YPD at OD 0.1
  3. Let the culture regrow to OD 1.3-1.5 (about 7 hours for me)
    • Meanwhile, if you have frozen Tris-DTT, thaw it on ice about an hour before you need it.
    • Also, chill your buffer E and the tubes holding the precipitated DNA
  4. Add 500uL Tris-DTT, return culture to incubator for 10-15 minutes
    • Place an aliquot of YPD in the incubator now (to warm it for the transformations)
  5. Spin down at 2500g, 4°C, 3min
  6. Wash with 25mL ice cold buffer E, then 1mL buffer E (decant supernatant, resuspend, respin).
  7. Resuspend in a final volume of 300uL ice cold buffer E (generally ~150-200uL buffer)
  8. Use 60uL to resuspend negative control DNA. Use remaining volume to resuspend remaining two tubes of DNA
  9. Transfer 50uL negative control mixture to chilled 2mm gap cuvette. Transfer 50uL transformation mix to each of 4 additional cuvettes.
  10. Electroporate at 540V, 25uF, infinite resistance, 2mm gap
    • After each transformation, resuspend cells with 1mL warm YPD. Transfer to a 15mL falcon tube. Then wash cuvette with another 1mL YPD and add that to the tube. All four positive transformations can go into the same falcon tube.
    • Time constant should be 15-20ms
  11. Incubate falcon tubes (shaking) for 1 hour
  12. Spin down (2500g, 4°C, 5 min) and resuspend in 1mL of appropriate dropout media
  13. Cultures can now be plated, or diluted (to >100mL) to grow in liquid media

Notes

  • In my hands, this gives 5x106 to 107 transformants total, with <103 background colonies (controls without insert or without vector)

References

For the original (more complete and detailed) description of the protocol:

  1. Chao G, Lau WL, Hackel BJ, Sazinsky SL, Lippow SM, and Wittrup KD. . pmid:17406305. PubMed HubMed [chao]

Contact

Josh Michener

Personal tools