Quick change Mutagenesis

Adapted from the stratagene QC protocol

 

 The two most important aspects of QC are the primers (design good ones) and the DNA. The DNA prep should be as clean as possible; promega midi preps followed my PCI-EtOH precipitation seems to work well.  Also make sure the DNA is purified from Dam+ cells, this means XL1-blue preferably, NOT BL21.

 

  1. Set up reactions as specified in the QC kit protocol start with a good amount of template DNA:

Ø      5 uL Pfu Turbo Buffer

Ø      1 uL 10 mM dNTPs

Ø      1.25 uL each primer at 100 ng/ul stock concentration

Ø      5 uL template DNA at 10 ng/uL

Ø      DH20 to a final volume of 49 uL

Ø      1 uL of Pfu Turbo Taq Polymerase (2.5 U/uL)

  1. Cycle as follows:

Ø      95 C for 30 seconds

Ø      95 C for 30 seconds

Ø      55 C of 1 minute

Ø      68 C for  ~ 2 minutes per Kb of plasmid length

Ø      Repeat steps 2-3 20-25 times

Ø      Hold at 4 C

  1. Remove reactions from thermocycler and add 1 uL of Dpn1 directly to the reaction mixture and incubate at 37 C for 1 hour. Note that a gel can easily be run at this stage to insure that products are present. Also if Wt plasmid persists in the reaction consider adding another 1 uL of Dpn1 after 1 hour and allowing the reaction to incubate at 37 C for an additional hour.
  2. Transform the reaction into XL1-blue competent cells.  Use 1 uL of the QC reaction per 50 uL of competent cells.  Incubate the mixture in a round bottom falcon culture tube for 30 minutes on ice and then heat shock the tube in the 42 C water bath for EXACTLY 45 seconds. Return the tube to the ice, add 500 uL of LB and outgrow the cells in the 37 C shaking incubator for 1 hour.
  3. Plate the transformation onto 2 plates of the appropriate selective media, 250 uL per plate.
  4. Allow the plates to dry thoroughly before flipping and incubate at 37 C overnight.
  5. IF colonies are present the next day they must be prepared for sequencing.  Pick an appropriate number of colonies and streak them onto another plate of the appropriate selective media.  Allow these to grow overnight at 37 C.
  6. Cell pop a single isolated colony form each of these overnights, making sure to note which plate the colony came from so you know EXACTLY which plate/colony the sequence will represent
  7. Clean up the PCR and have the product sequenced.
  8. If the sequence is correct then go back to the colony/plate that was sequenced.  You have your mutant!!! Do what you will with it. If it failed repeat steps 7 through 9 till you find a colony that’s correct.  Sometimes colonies from the same initial XL1 blue transformation will be wrong while others on the plate are correct.