Alice:LabNotes/2008-2-13: Difference between revisions

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Experiment: Repeat the padlock with control<br>
<b>Experiment: Repeat the padlock with control<br></b>
 
The experiment is repeated with the control sample from professor after ethanol preparation.<br>
The experiment is repeated with the control sample from professor after ethanol preparation.<br>


==Step 9:Page Denaturing Gel ==
Step 9:Page Denaturing Gel ==
1. Add 40 uL Novex TBE-Urea sample buffer (2x) for single stranded DNA to 40uL oligo mix.
  1. Add 40 uL Novex TBE-Urea sample buffer (2x) for single stranded DNA to 40uL oligo mix.
2. Mix 0.5 uL Invitrogen 10bp ladder with 9.5 uL dH2O, 10uL 2x TBE-Urea sample buffer.
  2. Mix 0.5 uL Invitrogen 10bp ladder with 9.5 uL dH2O, 10uL 2x TBE-Urea sample buffer.
3. Denature the DNA with loading dye on thermalcycler 75C for 15 min, then quickly transfer to ice, wait for 1 min.
  3. Denature the DNA with loading dye on thermalcycler 75C for 15 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.
  4. Use a 2D well denaturing gel, add 0.5x TBE buffer. Flush the wells very well to remove all urea.
6.Pre-run the gel for 10 min at 200V, load the samples into the wells, run at 200V for 30 min.
  6.Pre-run the gel for 10 min at 200V, load the samples into the wells, run at 200V for 30 min.
7. Stain gel with 5uL SYBR Gold in ~100 mL of TBE in clean tray. Place tray on an orbital shaker for ~5 min.
  7. Stain gel with 5uL SYBR Gold in ~100 mL of TBE in clean tray. Place tray on an orbital shaker for ~5 min.


== Step 9 continued: Purify the product ==
Step 9 continued: Purify the product ==
1. Prepare two gel shearing assemblies by placing a 0.5 mL tube within another 1.5 mL 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.
  Note: starting from this step, everything need to be done in the hood to avoid contamination.<br>
Note: Forgot to use Non-stick tube!
  1. Prepare two gel shearing assemblies by placing a 0.5 mL tube within another 1.5 mL 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 (85-92 nt in this case), chop the band into small slices, split into two half and transfer each half to one shearing assembly.
  Note: Forgot to use Non-stick tube!
3. Centrifuge at top speed > 12000 rpm for 2 min to sheer the gel, remove the 0.5 mL tube.
  2.Cut out the correct band (85-92 nt in this case), chop the band into small slices, split into two half and transfer each half to one shearing assembly.
4. Add 400 uL clean 1x TE buffer in the 1.5 mL tube. Tape the tubes on a vortexer, place the vortexer in 50C incubator, shake the tubes at low speed for 1 hour. <br>
  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 50C incubator, shake the tubes at low speed for 1 hour. <br>
  5. transfer the gel mix to Nanosep columns, spin for 3 min at top speed.<br>
  6. transfer supernatant to 1.5 ml tube. <br>


[[Image:ZhangLab_2 2008-02-13 13hr 47min.jpeg]]
[[Image:ZhangLab_2 2008-02-13 13hr 47min.jpeg]]
<br>
<br>
the above image is the 2D well gel I ran today, as we can see, there is no bright bands above the band i cut out, that means there are no cross or wrong annealing occurred, and the sample is quite purified.<br>
the above image is the 2D well gel I ran today, as we can see, there is no bright bands above the band i cut out, that means there are no cross or wrong annealing occurred, and the sample is quite purified.<br>

Latest revision as of 00:41, 8 March 2008

Experiment: Repeat the padlock with control

The experiment is repeated with the control sample from professor after ethanol preparation.

Step 9:Page Denaturing Gel ==

 1. Add 40 uL Novex TBE-Urea sample buffer (2x) for single stranded DNA to 40uL oligo mix.
 2. Mix 0.5 uL Invitrogen 10bp ladder with 9.5 uL dH2O, 10uL 2x TBE-Urea sample buffer.
 3. Denature the DNA with loading dye on thermalcycler 75C for 15 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.
 6.Pre-run the gel for 10 min at 200V, load the samples into the wells, run at 200V for 30 min.
 7. Stain gel with 5uL SYBR Gold in ~100 mL of TBE in clean tray. Place tray on an orbital shaker for ~5 min.

Step 9 continued: Purify the product ==

 Note: starting from this step, everything need to be done in the hood to avoid contamination.
1. Prepare two gel shearing assemblies by placing a 0.5 mL tube within another 1.5 mL 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. Note: Forgot to use Non-stick tube! 2.Cut out the correct band (85-92 nt in this case), chop the band into small slices, 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 50C incubator, shake the tubes at low speed for 1 hour.
5. transfer the gel mix to Nanosep columns, spin for 3 min at top speed.
6. transfer supernatant to 1.5 ml tube.

File:ZhangLab 2 2008-02-13 13hr 47min.jpeg
the above image is the 2D well gel I ran today, as we can see, there is no bright bands above the band i cut out, that means there are no cross or wrong annealing occurred, and the sample is quite purified.