Baylor-exon-Sept09 (11/4): Difference between revisions
>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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==Nicking Enzymes== | ==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''' | '''First Round''' | ||
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-10ul 10x NEBuffer2 | -10ul 10x NEBuffer2 | ||
-35ul H2O | -35ul H2O | ||
-3ul | -3ul Nt.A1wI enzyme | ||
Incubate at 37C for 2hrs -> 4C hold | Incubate at 37C for 2hrs -> 4C hold | ||
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*Run another Qiaquick purification | *Run another Qiaquick purification | ||
-> Elute with 50ul H2O | -> Elute with 50ul H2O | ||
==Size Selection== | ==Size Selection== | ||
Line 70: | Line 66: | ||
'''Preparation''' | '''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: | The ladders were created by adding: | ||
- | -1ul 10bp ladder | ||
-9ul H2O | -9ul H2O | ||
-10ul 2x TBU Buffer | -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 evident that the enzymatic digestion '''did not''' work: | 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''' | '''UV Cutting and Imaging''' |
Latest revision as of 23:42, 17 November 2009
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.
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[edit]
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