Quikchange (from primers to colonies!)

From 2010.igem.org

Contents

Primers

I use [http://mekentosj.com/enzymex/ enzyme x] to work with DNA sequences, it is installed on all the macs and has nice translation and reverse complement functions. Many of the same functions are available in [http://www.biology.utah.edu/jorgensen/wayned/ape/ a plasmid editor], which has Mac, Windows, and Linux versions.

  • Forward Primer
    • identify the bases that code for your residue of interest (eg. 295-297)
    • A - Copy the 10 bases before the codon (eg. 285-294)
    • B - Write your new codon after the 10 bases you just copied
    • C - Copy the 22 bases that follow the codon (eg. 298-319), I always terminate in a G or C
    • your forward primer should be 5'-A-B-C-3'
  • Reverse Primer
    • D - Copy the ten bases after the codon (eg. 298-307) - then reverse complement it!
    • E - Write your new codon - then reverse complement it!
    • F - Copy the 22 bases that precede the codon (eg. 273-294) - then reverse complement it!
    • your reverse primer should be 5'-D-E-F-3'
  • When they arrive make a 20uM stock

I use netprimer to make sure the primer Tm is good. You should have a rating above 70 (above 80 is preferable) for your primers.


PCR

Recipe

  • 1.25ul of 20uM Primer F
  • 1.25ul of 20uM Primer R
  • 10ul Phusion Buffer (5x)
  • 1ul of 10mM dNTPs
  • 0.5ul Template DNA (from miniprep, preferably from DH5alpha cells, but BL21 is still dam+ so it should be fine)
  • 1.0 ul Phusion polymerase
  • H2O to 50uL

Cycle

  • 98C for 30s
    • 98C for 5s
    • 53C for 20s
    • 72C for 20s/kb (usually plasmids are ~7kb = 2:20)
    • Cycle 16 times - more cycles are actually bad!
  • 72C for 8:00
  • 4C for hold

If you are using a different polymerase the annealing/extension/denaturing times and temperatures will be different!!

DpnI and transformation

  • After PCR, add 1ul of DpnI to each pcr tube
  • Incubate 1 hour-O/N at 37C
  • PCR Purify using Qaigen kit
  • Add 5ul of PCR reaction into 25ul DH5alpha cells
  • Incubate on ice for 5 minutes
  • Heatshock at 42C for 45 seconds
  • Recover on ice for 2 minutes
  • add 200ul LB (if in 96 well or 8 strip format) to each reaction
  • shake at 37C for 1 hour
  • plate on warmed antibiotic plates


If it doesn't work

  • Miniprep more colonies... ;)
  • order PAGE purified primers
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