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Team:Alberta/Notebook/Optimizations
From 2010.igem.org
Optimizations
PCR Using Universal Primers
Aug 27, 2010: Tested different amount of dNTPs added to 30uL PCR reactions. Results: Diluting the dNTPs to a final concentration of 0.2mM yielded cleaner PCR products. Aug 31, 2010: Experimented with the dNTP:MgCl2 ratios to see if it yielded cleaner and/or more PCR products while increasing the reaction volume to 50uL to increase the accuracy of measuring reagents. Results: Diluting MgCl2 to a final concentration of 2mM led to more specific PCR production without sacrificing too much of the quantity of desired products. Sept 1, 2010: Tested different annealing temperatures ranging from 55 to 70 degrees Celsius. Results: We did not find significant variation between the different annealing temperatures. We decided to go with 62 degrees Celsius since it yielded more products. Sept 2, 2010: Tested different cycles of 15, 20 and 25. Results: 25 Cycles produced a large enough quantity of PCR products for the amount of time required to run the program compared to the other cycles. Sept 10, 2010: Tested how the modification of using 1ng of template to 10ng will affect PCR efficiency. Results: No significance difference was seen.
BsaI Digestions
Sept 7, 2010: Tested different amounts of DNA samples – 0.5, 1, 1.5 and 2 ug – that can be digested by BsaI at 37oC and 50oC for one hour with and without BSA. Results: At 1.5 ug and up, the digestion is not cut to completion at 37oC. All samples are cut at 50oC without BSA. Sept 8, 2010: Tested how many ug of DNA could be digested in one hour with only one uL (10 units) of BsaI at 50oC. We used samples containing 2.5, 5 and 7.5ug of DNA. Results: At 5ug, the DNA is not completely cut, but majority of it is cut. Sept 10, 2010: Tested 2 to 4 hour digestion incubations at 37oC and 50oC with BsaI to see whether enzyme activity is maintained longer at 37oC. Results: BsaI enzymatic activity is maintained longer at 37oC while at 50oC BsaI enzymatic activity is lost in 2 hours. Sept 13, 2010: Test how long it will take to digest 5ug of DNA with 1uL BsaI at 50oC. Results: Loss of enzymatic activity in one hour. Test to see if 10ug of DNA will be digested overnight at 37oC (approximately 16 hours). Sept 14, 2010: Results: The DNA was not cut completely.
