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	*order PAGE purified primers		*order PAGE purified primers
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Latest revision as of 01:27, 23 October 2010

Contents 1 Primers 2 PCR 3 DpnI and transformation 4 If it doesn't work

Primers

We 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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