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[GUIDELINES]
<strong>Primers<br /></strong>I use 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 a plasmid editor , which has Mac, Windows, and Linux versions. Choose a codon for the new amino acid that is the most similar to the original codon. <br />&nbsp; &nbsp; &nbsp; -Forward Primer <br />&nbsp; &nbsp; &nbsp; &nbsp; &nbsp; -identify the bases that code for your residue of interest (eg. 295-297) <br />&nbsp; &nbsp; &nbsp; &nbsp; &nbsp; -A - Copy the 10 bases before the codon (eg. 285-294) <br />&nbsp; &nbsp; &nbsp; &nbsp; &nbsp; -B - Write your new codon after the 10 bases you just copied <br />&nbsp; &nbsp; &nbsp; &nbsp; &nbsp; -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' <br />&nbsp; &nbsp; &nbsp; -Reverse Primer <br />&nbsp; &nbsp; &nbsp; &nbsp; &nbsp; -D - Copy the ten bases after the codon (eg. 298-307) - <strong>then reverse complement it! </strong><br />&nbsp; &nbsp; &nbsp; &nbsp; &nbsp; -E - Write your new codon - <strong>then reverse complement it!</strong> <br />&nbsp; &nbsp; &nbsp; &nbsp; &nbsp; -F - Copy the 22 bases that precede the codon (eg. 273-294) - <strong>then reverse complement it!</strong> your reverse primer should be 5'-D-E-F-3' <br />&nbsp; &nbsp; &nbsp; -Order your primers from ElimBio (The newest PO B000177051) <br />&nbsp; &nbsp; &nbsp; -They should arrive in 200uM concentration, make a 20uM stock by diluting 5ul of Elim Primer with 45ul of dH2O&nbsp;<br /><br /><strong>PCR<br />Recipe<br /></strong>&nbsp; &nbsp; &nbsp; -Recipe 1.25ul of 20uM Primer F <br />&nbsp; &nbsp; &nbsp; -1.25ul of 20uM Primer R <br />&nbsp; &nbsp; &nbsp; -10ul Phusion Buffer (5x) 1ul of 10mM dNTPs <br />&nbsp; &nbsp; &nbsp; -0.5ul Template DNA (from miniprep, preferably from DH5alpha cells, but BL21 is still dam+ so it should be fine - 5ng starting template is plenty! (note: good range = 50 - 100 ng) ) <br />&nbsp; &nbsp; &nbsp; -0.5ul Phusion polymerase <br />&nbsp; &nbsp; &nbsp; -35ul H20 <br /><strong>Cycle</strong> <br />&nbsp; &nbsp; &nbsp; -98C for 30s 98C for 5s (I usually do this step for 10 seconds) <br />&nbsp; &nbsp; &nbsp; -53C for 20s <br />&nbsp; &nbsp; &nbsp; -72C for 20s/kb (usually plasmids are ~7kb = 2:20)<br />&nbsp; &nbsp; &nbsp; &nbsp; &nbsp; -Cycle 16 times - <em>more cycles are actually bad!</em> <br />&nbsp; &nbsp; &nbsp; -72C for 8:00 <br />&nbsp; &nbsp; &nbsp; -4C for hold<strong><br /><br /><br /></strong><strong>If it does not work:<br /></strong>&nbsp; &nbsp; &nbsp; -Miniprep more colonies... ;) <br />&nbsp; &nbsp; &nbsp; -Order PAGE purified primers if you haven't already. We have a good price from Invitrogen. <br />&nbsp; &nbsp; &nbsp; -Redesign your primers with netprimer <br />&nbsp; &nbsp; &nbsp; -For high background of WT sequence, decrease starting amount of template (really, 5ng is plenty), &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; -Get fresh Dpn1 and digest ~24hrs. <br />&nbsp; &nbsp; &nbsp; -Run a gradient of annealing temperatures (try 52-62).<br /><br />Split reaction protocol (for high primer-dimer problems): <br /><strong>Recipe F</strong> <br />&nbsp; &nbsp;-1ul of 20uM Primer F <br />&nbsp; &nbsp;-5ul Phusion Buffer (5x) <br />&nbsp; &nbsp;-0.5ul of 10mM dNTPs <br />&nbsp; &nbsp;-0.5ul Template DNA (from miniprep, preferably from DH5alpha cells, but BL21 is still dam+ so it should be fine) 0.5ul Phusion polymerase <br />&nbsp; &nbsp;-17.5 ul H20 <br /><strong>Recipe R</strong> <br />&nbsp; &nbsp;-1ul of 20uM Primer F <br />&nbsp; &nbsp;-5ul Phusion Buffer (5x) <br />&nbsp; &nbsp;-0.5ul of 10mM dNTPs <br />&nbsp; &nbsp;-0.5ul Template DNA (from miniprep, preferably from DH5alpha cells, but BL21 is still dam+ so it should be fine)<br />&nbsp; &nbsp;-0.5ul Phusion polymerase <br />&nbsp; &nbsp;-17.5 ul H20<br /><br /><strong>Cycle each F and R in separate tubes for:</strong> <br />&nbsp; &nbsp;-98C for 30s <br />&nbsp; &nbsp; &nbsp; &nbsp; &nbsp; -98C for 5s (I usually do this step for 10 seconds)<br />&nbsp; &nbsp; &nbsp; &nbsp; &nbsp; -53C for 20s <br />&nbsp; &nbsp; &nbsp; &nbsp; &nbsp; -72C for 20s/kb (usually plasmids are ~7kb = 2:20) <br />&nbsp; &nbsp; &nbsp; &nbsp; &nbsp; -Cycle 10 times <br />&nbsp; &nbsp;-72C for 8:00 <br />&nbsp; &nbsp;-4C for hold<br /><br /><strong>Mix tubes F and R and cycle: </strong><br />&nbsp; &nbsp;-98C for 30s <br />&nbsp; &nbsp; &nbsp; &nbsp; &nbsp; -98C for 5s (I usually do this step for 10 seconds) <br />&nbsp; &nbsp; &nbsp; &nbsp; &nbsp; -53C for 20s <br />&nbsp; &nbsp; &nbsp; &nbsp; &nbsp; -72C for 20s/kb (usually plasmids are ~7kb = 2:20) <br />&nbsp; &nbsp; &nbsp; &nbsp; &nbsp; -Cycle 10 times <br />&nbsp; &nbsp;-72C for 8:00 <br />&nbsp; &nbsp;-4C for hold <br /><br />Follow transformation, DpnI digest as above<br /> <br /><strong>Degenerator</strong>: http://andersonlab.qb3.berkeley.edu/Software/Degenerator.html <br /><br /> NB: Not to be confused with this:<a href="http://www.imdb.com/title/tt0100449/" target="_blank"> http://www.imdb.com/title/tt0100449/ </a><br /><br /><br />

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