Baylor-exon-Sept09: Difference between revisions

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(New page: '''Oligo Separation''' 1x 48x Template 0.4ul 19.2ul 2x Kapa Master Mix 50ul 50ul...)
 
>Jbrubake
 
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'''Oligo Separation'''
===Baylor (BCM) Creation===


                                          1x              48x
'''PCR'''
Template                         0.4ul         19.2ul
                          1x              48x
2x Kapa Master Mix       50ul            50ul
Template               0.4ul           19.2ul  
H2O                                 50ul            50ul
2x Kapa Master Mix     50ul            50ul
eMIP-CA1F (100uM)     0.4ul           0.4ul
H2O                     50ul            50ul
eMIP-CA1R (100uM)     0.4ul         0.4ul
eMIP-CA1F (100uM)     0.4ul           0.4ul
'''PCR:''' 95C, 30sec -> (95C, 3sec -> 54C, 30sec -> 60C, 20sec -> plate read) x 25 -> 60C, 2min -> 15C hold
eMIP-CA1R (100uM)     0.4ul           0.4ul
95C, 30sec -> (95C, 3sec -> 54C, 30sec -> 60C, 20sec -> plate read) x 25 -> 60C, 2min -> 15C hold


*Note: Amplification started after ~13 cycles
*Note: Amplification started after ~13 cycles
'''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
==Nicking Enzymes==
'''First Round'''
For each 50ul sample, add:
-10ul  10x NEBuffer2
-35ul  H2O
-3ul    Nt.A1wI enzyme
Incubate at 37C for 2hrs -> 4C hold
*Run another Qiaquick purification
'''Second Round'''
For each 50ul sample, add:
-2ul Nb.BsrDI enzyme
Incubate at 65C for 2hrs -> 4C hold
*Run another Qiaquick purification
-> Elute with 50ul H2O
'''Nanodrop Readings'''
BCM Tube 1: 143.8 ng/ul * 50ul = '''7.2ug'''
BCM Tube 2: 163.5 ng/ul * 50ul = '''8.2ug'''
*Note: Split each tube into 3 separate tubes for size selection, making 6 tubes total so as not to overload each gel.
==Size Selection==
'''Preparation'''
Due to high concentration, each tube of 50ul was split into 3 tubes of ~16.3ul. 32.7ul of H2O was added to make a total volume of 50ul per tube. 50ul of 2x TBU Buffer was then added and the tubes were vortexed and spun down.
The ladders were created by adding:
-1.5ul 10bp ladder
-9ul H2O
-10ul 2x TBU Buffer
Once the TBU gel was prepped, the samples were incubated at 75C for ~7min, then put on ice for ~1min. The ladders and samples were then loaded into the gel. After running the gels for 20min at 250V, they were stained with SYBR Gold. Upon viewing the gels through a UV stage, it was evident that the enzymatic digestion '''did not''' work:
'''UV Cutting and Imaging'''
[[Image:BCM_SizeSelection_1.jpg]]
''Gel Image 1''
[[Image:BCM_SizeSelection_2.jpg]]
''Gel Image 2''

Latest revision as of 09:17, 7 November 2009

Baylor (BCM) Creation[edit]

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
  • Note: Amplification started after ~13 cycles

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

Nicking Enzymes[edit]

First Round

For each 50ul sample, add:
-10ul   10x NEBuffer2
-35ul   H2O
-3ul     Nt.A1wI enzyme
Incubate at 37C for 2hrs -> 4C hold
  • Run another Qiaquick purification

Second Round

For each 50ul sample, add:
-2ul Nb.BsrDI enzyme
Incubate at 65C for 2hrs -> 4C hold
  • Run another Qiaquick purification

-> Elute with 50ul H2O

Nanodrop Readings

BCM Tube 1: 143.8 ng/ul * 50ul = 7.2ug
BCM Tube 2: 163.5 ng/ul * 50ul = 8.2ug
  • Note: Split each tube into 3 separate tubes for size selection, making 6 tubes total so as not to overload each gel.

Size Selection[edit]

Preparation

Due to high concentration, each tube of 50ul was split into 3 tubes of ~16.3ul. 32.7ul of H2O was added to make a total volume of 50ul per tube. 50ul of 2x TBU Buffer was then added and the tubes were vortexed and spun down.

The ladders were created by adding:

-1.5ul 10bp ladder
-9ul H2O
-10ul 2x TBU Buffer

Once the TBU gel was prepped, the samples were incubated at 75C for ~7min, then put on ice for ~1min. The ladders and samples were then loaded into the gel. After running the gels for 20min at 250V, they were stained with SYBR Gold. Upon viewing the gels through a UV stage, it was evident that the enzymatic digestion did not work:

UV Cutting and Imaging

File:BCM SizeSelection 1.jpg

Gel Image 1

File:BCM SizeSelection 2.jpg

Gel Image 2