Team:Alberta/Notebook/Optimizations

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TEAM ALBERTA

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.


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