#SiteDirectedMutagenesis is one of the easiest ways to manipulate gene sequences mostly effective but not limited to #PlasmidDNA. It empowers a #MolecularBiologist in more than one ways to play around #sequences and #structures of #nucleicacids and #proteins by means of #PCR and #RestrictionEndonucleases. Dive in for essential tips!
Suggestions:
#Gradient Ta PCR: This is fairly simple yet effective. Set up a 120uL reaction and divide it into 12 tubes of 10uL each, spread across the 12 different annealing temperature (#Ta). DpnI treat the reaction and transform 5uL of it in 50uL cells.
#PCRAdditives: Use of 3% #DMSO, 2-3M #Betaine helps in resolving secondary structures of the #templateDNA, as well as making it easier to proceed with long #primers, typical of #SDM reactions.
Use suitable cells: #TakaraDH5alpha cells work much better than #AgilentXL10GoldUltra competent cells. Also it is suggested to use <=10% volume of reaction mix while transforming cells.
#Decrease Extension temperature: Since SDM reactions produce the whole plasmid, extended time at 72C might degrade or affect the #DNApolymerase. So it is advised to bring it down to 68C for products >3kb.
#Gradient template DNA PCR: Using a varied amount of template DNA also proves good at times.
#Dialyze reaction to get rid of excess salts, DMSO and other additives, which might affect the #TransformationEfficiency.
Is there something i missed? Please do mention in the comments.
Happy mutagenesis!
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