Enzyme Test: Difference between revisions

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>Jbrubake
(New page: *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...)
 
>Jbrubake
Line 2: Line 2:


==BCM Preparation==
==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


==Nt.A1wI==
==Nt.A1wI==

Revision as of 22:23, 20 November 2009

  • 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

Nt.A1wI