---+ Site-directed mutagenesis protocol (adapted from QuickChange) A protocol for changing one (or a few) bases on a plasmid ---++ SUPPLIES: ---+++ Primer Design: Use the following website to design the QuickChange primers: </br> http://www.bioinformatics.org/primerx/cgi-bin/DNA_1.cgi ---+++ Equipment: * Thermocycler * 37°C Incubator ---+++ Consumables: * PCR tubes * Reagents in table below * Dpn1 restriction enzyme (to remove template from rnx) ---+++ Buffers and Solutions: <b>QuickChange rxn: </b> | *Reagent* | *Amount / L* | | DNA template (~5ng/µl) | 2.0 µl | | 5x HF buffer | 10 µl | | 10 mM dNTPs | 1 µl | | 10µM Forward Primer | 2.5 µl | | 10µM Forward Primer | 2.5 µl | | 100% DMSO | 2.5 µl | | 50 mM MgCl<sub>2</sub> | 1 µl | | Phusion Polymerase | 0.5 µl | | H<sub>2</sub>O | 28 µl | | <b>TOTAL</b>| <b>50 µl</b> | ---++ PROTOCOL Thermocycler settings: 1) 98C 30 sec 2) 98C 30 sec 3) 55C 1 min (can be empirically optimized) 4) 72F 1 min/kb (can change time for your polymerase of choice) * repeat steps 2-4 x12-18 5) hold at 10C add 1µl Dpn1 to each tube and mix thoroughly. Incubate for 1hr at 37C (thermocycler or incubator). Proceed to transformation or store at -20C. -- Main.MattMcGuffie - 25 Oct 2018
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Topic revision: r1 - 2018-10-25 - MattMcGuffie