Baylor-exon-Sept09 (11/19)
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.
I decided to run a NanoDrop on the product so that I could more thorougly calibrate the enzymes. Reading: 313.9ng/ul * 70ul sample = 22ug DNA for digestion
First Round
For each 50ul sample, add: -6ul 10x NEBuffer2 -2ul Nt.A1wI enzyme Incubate at 37C for 2hrs -> 4C hold
- Run another Qiaquick purification
Second Round
For each 50ul sample, add: -6ul 10x NEBuffer2 -2ul Nb.BsrDI enzyme Incubate at 65C for 2hrs -> 4C hold
- Run another Qiaquick purification
-> Elute with 50ul H2O
Size Selection
Preparation
NanoDrop readings:
Tube I: 112ng/ul * 50ul = 5.6ug Tube II: 111ng/ul * 50ul = 5.6ug
Because of the high amount of DNA I split each tube into 2x 25ul tubes and added 25ul 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. Upon viewing the gels through a UV stage, it was again evident that the enzymatic digestion did not work:
UV Cutting and Imaging
Gel Image 1
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.
Quantification
Each sample was quantified via two methods. First I used a Qubit Flourometer, then two different gel quantifications were run. To be safe, the conservative concentrations were used.
Results (all in ng/ul): Qubit Gel 1 Gel 2
bis 30.8 13.5 CES 32.5 14.0 BCM 3.84 4.75