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 ==
Note: starting from this step, everything need to be done in the hood to avoid contamination.<br>
  Note: starting from this step, everything need to be done in the hood to avoid contamination.<br>
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.
  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!
  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.
  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.
  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>
  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.