Enzyme Test
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- Both rounds of Nicking Enzymes, used on both BCM sets, failed to properly digest the DNA. In order to get a better understanding of the situation, the BCM set was re-amplified and the two enzymes, Nt.A1wI and Nb.BsrDI were tested as follows:
BCM Preparation
PCR
1x 48x Template 0.4ul 19.2ul 2x Kapa Master Mix 50ul 50ul H2O 50ul 50ul eMIP-CA1F (100uM) 0.4ul 0.4ul eMIP-CA1R (100uM) 0.4ul 0.4ul 95C, 30sec -> (95C, 3sec -> 54C, 30sec -> 60C, 20sec -> plate read) x 25 -> 60C, 2min -> 15C hold
Ethanol Precipitation
Collected 24 reactions in a 16mL tube and added: 0.1x 3M NaOAc (240ul) 2.5x 100% EtOH (6mL) 8ul GlycoBlue -> Allow to cool in -70C freezer for at least 20 minutes -> Spin in 4C at 4k rpm for 20min -> Remove all liquid -> Suspend palette in 750ul 75% EtOH -> Move solution from 16mL tube to fresh 1.6mL tube -> Spin in 4C at 10k rpm for 5min -> Remove all liquid, allow palette to dry -> Suspend palette in 100ul H2O
Qiaquick Purification
Per tube of oligo solution: -Add 5x PB buffer -Mix in 1.6mL tubes and transfer solution to Qiaquick columns -Spin at 14k rpm for 1min -Toss supernatant -Add 750ul PE buffer to column -Spin at 14k rpm for 1min -Toss supernatant -Spin again, toss collection tube -Transfer column to fresh 1.6mL tube -Elute with 50ul H2O per column -Spin at 14k rpm for 1:30 -Toss Qiaquick column
- Note: After the QIAquick purification, I attempted to use the NanoDrop to roughly estimate the amount of DNA. Unfortunately, I was unable to acquire any data as the NanoDrop machine was unable to process the sample. A gel was run to ensure that there was in fact sample in the mixture, which the gel confirmed:
From here 20ul were taken from the main tube of BCM solution (which originally contained 100ul) and were split into 2 tubes of 10ul solution. To each tube, 40ul water was added to make a total volume of 50ul for each tube.
Nt.A1wI
Using one of the 50ul tubes, I added:
10ul 2x NEBuffer2 35ul H2O 3ul Nt.A1wI enzyme
The reagents were incubated for 2 hours at 37C
Nb.BsrDI
Using the other 50ul tube, I added:
2ul Nb.BsrDI enzyme
The reagents were incubated for 2 hours at 65C
Results
After running both enzymes through size-selection gels, it is clear that the Nb.BsrDI enzyme is NOT working properly: