BisFirstExon: Difference between revisions

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'''Nanodrop Readings'''
'''Nanodrop Readings'''
  CES Tube 1: 169.3 ng/ul * 50ul = 8.5ug
  bis Tube 1: 170.2 ng/ul * 50ul = 8.5ug
  CES Tube 2: 148.7 ng/ul * 50ul = 7.4ug
  bis Tube 2: 178.6 ng/ul * 50ul = 8.9ug


==Size Selection and Extraction==
==Size Selection and Extraction==

Revision as of 21:26, 6 November 2009

bisFirstExon (bis) Creation

PCR

                         1x               48x
Template               0.4ul           19.2ul 
2x Kapa Master Mix      50ul             50ul
H2O                     50ul             50ul
AP1V41U (100uM)        0.4ul            0.4ul
AP2V4 (100uM)          0.4ul            0.4ul
95C, 30sec -> (95C, 3sec ->  60C, 50sec -> plate read) x 25 -> 60C, 2min -> 15C hold
  • Note: Amplification stated at ~15 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

Enzymatic Digestion

DpnII Enzyme

For each 50ul sample, add:
-2ul DpnII (50U/ul) enzyme
-5ul DpnII 10x Buffer
-Incubate at 37C for 2hrs -> 4C hold

USER Enzyme

For each ~60ul sample, add:
-4ul USER enzyme
-Incubate at 37C for 2hrs -> 4C hold
  • Run another Qiaquick purification

Nanodrop Readings

bis Tube 1: 170.2 ng/ul * 50ul = 8.5ug
bis Tube 2: 178.6 ng/ul * 50ul = 8.9ug

Size Selection and Extraction

Size Selection

These samples were split up into 6 tubes total, making volumes:

16.3ul bis Sample
33.7ul H2O
50ul 2x TBU Buffer
Total volume: 100ul

The samples were run against a 10bp ladder. Each gel was then cut to excise the band at ~100bp. All three sets of gels were fairly clean; I encountered no problems:

File:Bis sizeselection 1

First set of bis gels

File:Bis sizeselection 2

Second set of bis gels

File:Bis sizeselection 3

Third set of bis gels

After excising the correct band from the gel, each band was cut in half. These halves were placed into shearing tubes (a 0.5mL tube with a hold poked through the bottom resting freely inside a 1.6mL tube) and spun down at 14,000 rpm for 1:30.

Extraction

After the gel fragments were sheared, the 0.5mL tubes were tossed and 400ul of 1x TE Buffer was added to each tube. Each tube was then placed in a 37C incubator and placed on a spinner/vortexer for approximately 1 hour. After this each tube was run through another round of ethanol purification.