TEAM ALBERTA

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Optimizations

Project Timeline: Click on an image to see more information

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^oC to 70^oC.

Results: We did not find significant variation between the different annealing temperatures. We decided to go with 62^oC 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 – 0.5ug, 1ug, 1.5ug and 2ug – that can be digested by BsaI at 37^oC and 50^oC for one hour with and without BSA.

Results: At 1.5 ug and up, the digestion is not cut to completion at 37^oC. All samples are cut at 50^oC 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 50^oC. We used samples containing 2.5ug, 5ug 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 37^oC and 50^oC with BsaI to see whether enzyme activity is maintained longer at 37^oC.

Results: BsaI enzymatic activity is maintained longer at 37^oC while at 50^oC 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 50^oC.

Results: Loss of enzymatic activity in one hour.

Test to see if 10ug of DNA will be digested overnight at 37^oC (approximately 16 hours).

• Sept 14, 2010:

Results: The DNA was not cut completely.