Baylor-exon-Sept09

From ZhangLabWiki
Revision as of 23:56, 4 November 2009 by >Jbrubake (→‎Size Selection)
Jump to navigation Jump to search

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

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: -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 evident that the enzymatic digestion did not work:

UV Cutting and Imaging


Gel Image 1

Gel Image 2