Alice:LabNotes/2008-3-13

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Experiment 1: Padlock library 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.
 Note: the samples used to run is the gel is the remaining 2/3 (~20 uL).

File:ZhangLab 2 2008-03-13 17hr 56min.jpeg


Result: two bands were cut out from the gel since the gel doesn't seem to be very straight, and there were two bright bands close to the 80 - 90 ng ladder bands.

Step 9 continued: Purify the product

 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 (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 overnight.
 
 5. transfer the gel mix to Nanosep columns, spin for 3 min at top speed.
 
 6. transfer supernatant to 1.5 ml tube.