Baylor-exon-Sept09 (11/28)

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Revision as of 22:24, 1 December 2009 by >Jbrubake (New page: ===Baylor (BCM) Creation=== '''PCR''' 1x 48x Template 0.4ul 19.2ul 2x Kapa Master Mix 50ul 50ul H2O ...)
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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:
-6ul   10x NEBuffer2
-5ul    Nt.A1wI enzyme
Incubate at 37C for 2hrs -> 4C hold
  • Run another Qiaquick purification

Second Round

For each 50ul sample, add:
-6ul 10x NEBuffer2
-5ul Nb.BsrDI enzyme
Incubate at 65C for 2hrs -> 4C hold
  • Run another Qiaquick purification

-> Elute with 50ul H2O

Size Selection

Preparation

I split each sample in half (50ul -> 2x 25ul) and filled the remaining volume (25ul) with water. 50ul of 2x TBU Buffer was then added.

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.

UV Cutting and Imaging

File:11/28 BCM 1.jpg

Gel Image 1

File:11/28 BCM 2.jpg

Gel Image 2

Extraction

I excised the bands at 100bp and sheared them at 14k rpm for 1:30. 400ul 1x TE solution was then added to each tube (8 tubes total) of sheared gel and the resulting mixture was incubated/vortexed at 37C for about 1 hour.

The liquid remains were spun through a NanoSep column, which was then put through ethanol purification. Each tube was then eluted with 5ul water. When pooled, the total volume of BCM sample was roughly 40ul.

I took this new (11/28) BCM sample and added it to the previous (11/19) BCM sample. I then ran another QIAquick purification to combine all the product and augment the concentration. The final product was eluted with 24ul water (enough to run multiple quantifications on).

Quantification

The sample was quantified via three methods. First I used a Qubit Flourometer, then a NanoDrop reading, then a gel quantification was run. To be safe, the conservative concentration was used.

Results (all in ng/ul):
                  Qubit             NanoDrop               Gel         
BCM               40.0              45.0                15.2

Using the gel quantification, the final product was 20ul with a concentration of 15ng/ul