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==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 [[Category:Protocol]]

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