Baylor-exon-Sept09 (11/28): Difference between revisions
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'''UV Cutting and Imaging''' | '''UV Cutting and Imaging''' | ||
[[Image: | [[Image:11_28_BCM_1.jpg]] | ||
''Gel Image 1'' | ''Gel Image 1'' | ||
[[Image: | [[Image:11_28_BCM_2.jpg]] | ||
''Gel Image 2'' | ''Gel Image 2'' | ||
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==Quantification== | ==Quantification== | ||
Gel image: | |||
[[image: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. | 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): | Results (all in ng/ul): | ||
Qubit NanoDrop | Qubit NanoDrop Gel | ||
BCM 40.0 45.0 15.2 | BCM 40.0 45.0 15.2 | ||
Using the gel quantification, the final product was 20ul with a concentration of 15ng/ul | Using the gel quantification, the final product was 20ul with a concentration of 15ng/ul |
Latest revision as of 22:29, 1 December 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
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
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.
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:
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