Alice:LabNotes/2008-2-7
Experiment 1: Padlock library preparation
Step 8: Purify the reaction with Qiagen MinElute columns
1. Add 750 uL of PBI buffer, mix well, load around 450 uL to each column (2 columns with total of 900 uL)
2. Spin at top speed (14000 rpm) for 1 min
3. Empty the collection tube, add 750 uL wash buffer to the column
4. Spin at top speed for 1 min
5. Empty the collection tube, spin at top speed for 1 min
6. Carefully transfer the columns to clean 1.6 mL tubes
7. Place the column/tube assemblies to 37C incubator, incubate for 5 min
8. Add 20 uL EB buffer to each column, wait for 1 min, spin at top speed for 1 min.
9. Pool DNAs from the two tubes into one, measure the concentration with Nanodrop.
Nanodrop result:
sample type:ssDNA-33
260/280: 1.85
260/230: 1.96
62.7 ng/uL
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.
File:ZhangLab 2 2008-02-07 14hr 11min.jpeg
Note: starting from this step, everything need to be done in the hood to avoid contamination.
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: 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.