Alice:LabNotes/2008-9-15: Difference between revisions
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>Zsakura2 |
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6. Stain gel with 5uL SYBR Gold in ~100 mL of TBE in clean tray. Place tray on an orbital shaker for ~5 min. | 6. Stain gel with 5uL SYBR Gold in ~100 mL of TBE in clean tray. Place tray on an orbital shaker for ~5 min. | ||
Purify the product from the gel | 2D gel to take out the band we are interestd in (160bp): | ||
[[Image:ZhangLab_2 2008-09-15 12hr 53min.jpg]] | |||
Purify the product from the gel | |||
1. Prepare two gel shearing assemblies by placing a 0.5 mL tube within another 1.5 mL Non-Stick tube. Use | 1. Prepare two gel shearing assemblies by placing a 0.5 mL tube within another 1.5 mL Non-Stick tube. Use | ||
G20 needle to punch a small hole at the bottom of the 0.5 mL tube. You need two assemblies for each PAGE gel. | G20 needle to punch a small hole at the bottom of the 0.5 mL tube. You need two assemblies for each PAGE gel. | ||
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5. In the PCR hood, transfer the gel mix to Nanosep columns, spin for 3 min at top speed (15,000 rpm), and then transfer | 5. In the PCR hood, transfer the gel mix to Nanosep columns, spin for 3 min at top speed (15,000 rpm), and then transfer | ||
supernatant to 1.5mL tube. | supernatant to 1.5mL tube. | ||
Revision as of 20:02, 15 September 2008
extending the probe length
After PCR amplification after CircleHelper insertion, T7 exonuclease digestion was performed. And the detailed procedures can be found through this link: [[1]]
Page Denaturing Gel 1. Add TBE-Urea sample buffer (2x) in each tube. 2. Mix 0.5 uL Invitrogen 10bp ladder with 3.5 uL dH2O, 4 uL 2x TBE-Urea sample buffer. 3. Denature at 75C for 8 min, then quickly transfer to ice, wait for 1 min. 4. Use a 2D well denaturing gel, add 0.5x TBE buffer. Flush the wells very well to remove all urea. 5. Pre-run the gel for 10 min at 200V, load the samples into the wells, run at 200V for 30 min. 6. Stain gel with 5uL SYBR Gold in ~100 mL of TBE in clean tray. Place tray on an orbital shaker for ~5 min. 2D gel to take out the band we are interestd in (160bp): File:ZhangLab 2 2008-09-15 12hr 53min.jpg
Purify the product from the gel 1. Prepare two gel shearing assemblies by placing a 0.5 mL tube within another 1.5 mL Non-Stick tube. Use G20 needle to punch a small hole at the bottom of the 0.5 mL tube. You need two assemblies for each PAGE gel. 2.Cut out the correct band, split into two half and transfer each half to one shearing assembly. 3. Centrifuge at top speed > 12000 rpm for 2 min to sheer the gel, remove the 0.5 mL tube. 4. Add 400 uL clean 1x TE buffer in the 1.5 mL tube. Tape the tubes on a vortexer, place the vortexer in 37C incubator, shake the tubes at low speed for 45 min. 5. In the PCR hood, transfer the gel mix to Nanosep columns, spin for 3 min at top speed (15,000 rpm), and then transfer supernatant to 1.5mL tube.