Baylor-exon-Sept09 (11/4): Difference between revisions

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(New page: ===Baylor (BCM) Creation=== '''PCR''' 1x 48x Template 0.4ul 19.2ul 2x Kapa Master Mix 50ul 50ul H2O ...)
 
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==Nicking Enzymes==
==Nicking Enzymes==
*NOTE: These enzymes were ''fresh'', having arrived only on 11/6/09. However, I was not able to proceed with the enzymatic digestion until 11/15/09.


'''First Round'''
'''First Round'''
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*Run another Qiaquick purification
*Run another Qiaquick purification
-> Elute with 50ul H2O
-> 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==
==Size Selection==
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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.
I decided to skip the NanoDrop quantification and split each tube in half, yielding 4x 25ul samples. Added 25ul water and 50ul 2x TBU Buffer to each tube, making a total volume of 100ul.


The ladders were created by adding:
The ladders were created by adding:
  -1.5ul 10bp ladder
  -1ul 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 '''again''' evident that the enzymatic digestion '''did not''' work:


'''UV Cutting and Imaging'''
'''UV Cutting and Imaging'''

Revision as of 23:42, 17 November 2009

Baylor (BCM) Creation

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

  • NOTE: These enzymes were fresh, having arrived only on 11/6/09. However, I was not able to proceed with the enzymatic digestion until 11/15/09.

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

Size Selection

Preparation

I decided to skip the NanoDrop quantification and split each tube in half, yielding 4x 25ul samples. Added 25ul water and 50ul 2x TBU Buffer to each tube, making a total volume of 100ul.

The ladders were created by adding:

-1ul 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 again evident that the enzymatic digestion did not work:

UV Cutting and Imaging

File:BCM 2 SizeSelection 1.jpg

Gel Image 1

File:BCM 2 SizeSelection 2.jpg

Gel Image 2: Extracted band at 120bp

  • Note: I decided to continue with half of the gel content excised at 120 to be compared with the CES and bis sets