Baylor-exon-Sept09 (11/28)

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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

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:
-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[edit]

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[edit]

Gel image:

File:BCM Quantification.jpg

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