Enzyme Test

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Revision as of 22:51, 20 November 2009 by >Sam Chiang (→‎Nb.BsrDI)
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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:

File:3 sets.jpg


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