Baylor-exon-Sept09: Difference between revisions

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'''Preparation'''
'''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.
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:
The ladders were created by adding:
-1ul 10bp ladder
-1.5ul 10bp ladder
-9ul H2O
-9ul H2O
-10ul 2x TBU Buffer
-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:
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''
''Gel Image 1''
[[Image:BCM_SizeSelection_2.jpg]]


''Gel Image 2''
''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