PCR技术(七)资料.ppt

* QUESTIONS 9 Short PCR products in my multiplex reaction are weak. How can I improve their yield? Increase KCl (buffer) concentration to 1.2x-2x, but keep MgCl2 concentration at 1.5-2mMDecrease denaturing timeDecrease annealing time and temperatureDecrease extension time and temperatureIncrease amount of primers for the weak loci while decreasing the amount for the strong loci.Add adjuvants. Best, use BSA (0.1 to 0.8 μg/μL final concentration). You can also try 5% (v/v, final concentration) DMSO or glycerolCombine some/all of the above  * QUESTIONS 10 Longer PCR products in my multiplex reaction are weak. How can I improve their yield?  Decrease KCl (buffer) concentration to 0.7-0.8x, but keep MgCl2 concentration at 1.5-2mMIncrease MgCl2 concentration up to 3-4.5 mM but keep dNTP concentration constant.Increase denaturing timeIncrease annealing timeDecrease annealing temperatureIncrease extension time and temperatureIncrease amount of primers for the weak loci while decreasing the amount for the strong lociAdd adjuvants. Best, use BSA (0.1 to 0.8 μg/μL final concentration). You can also try 5% (v/v, final concentration) DMSO or glycerolCombine some/all of the above * QUESTIONS 11 All products in my multiplex reaction are weak. How can I improve the yield? Decrease annealing time in small steps (2o C)Decrease extension temperature to 62-68o CIncrease extension timeIncrease template concentrationIncrease overall primer concentrationAdjust Taq polymerase concentrationChange KCl (buffer) concentration, but keep MgCl2 concentration at 1.5-2mMIncrease MgCl2 concentration up to 3-4.5 mM but keep dNTP concentration constant.Add adjuvants. Best, use BSA (0.1 to 0.8 μg/μL final concentration). You can also try 5% (v/v, final concentration) DMSO or glycerolCombine some/all of the above * QUESTIONS 12Unspecific products appear in my multiplex reaction. Can I get rid of them somehow? If long: increase buffer concentration to 1.2-2x, but keep Mg