Baylor-exon-Sept09 (11/4)
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.
First Round
For each 50ul sample, add: -10ul 10x NEBuffer2 -35ul H2O -3ul Nt.A1wI enzyme Incubate at 37C for 2hrs -> 4C hold
- Run another Qiaquick purification
Second Round
For each 50ul sample, add: -2ul Nb.BsrDI enzyme Incubate at 65C for 2hrs -> 4C hold
- Run another Qiaquick purification
-> Elute with 50ul H2O
Size Selection
Preparation
I decided to skip the NanoDrop quantification and split each tube in half, yielding 4x 25ul samples. Added 25ul water and 50ul 2x TBU Buffer to each tube, making a total volume of 100ul.
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
File:BCM 2 SizeSelection 1.jpg
Gel Image 1
File:BCM 2 SizeSelection 2.jpg
Gel Image 2: Extracted band at 120bp
- Note: I decided to continue with half of the gel content excised at 120 to be compared with the CES and bis sets