Baylor-exon-Sept09 (11/19)
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
- 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[edit]
- 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[edit]
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.
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[edit]
Gel Images:
File:Bis BCM CES Quantification1.jpg
Gel image 1
File:Bis BCM CES Quantification2.jpg
Gel image 2
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 Average (Between Gels) bis 30.8 6.62 4.54 5.60 CES 32.5 6.89 4.40 5.60 BCM 3.84 2.9 1.26 2.10