Alice:LabNotes/2008-4-1

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Experiment 1: Padlock Library Preparation continued

 Step 11: Quantification of DNA using denaturing gel and a quantitative ladder
1. Add 8uL of 100bp ladder and 8 uL of 2x TBE-Urea buffer
2. Dilute sample 10x (2uL sample + 8 uL of water) so total is 10uL of sample, and add 10 uL of 2x TBE-Urea buffer.
3. Denature the DNA with loading dye on thermalcycler 75C for 5 min, then quickly transfer to ice, wait for 1 min.
Note: Transfer to ice to keep the samples from further denaturation.
4. Use 6% 10 well TBU gel, add 0.5x TBE buffer, flush the wells very well to remove all urea.
5. Pre-run the gel at 200V for 10 min, then load the samples into the wells, run at 200V for 30 min. Load the ladder as 8 uL, and load the samples as 10, 5 uL.
6. Stain the gel with 5 uL SYBR gold in ~100 mL of TBE in clean tray. Place the tray on an orbital shaker for 5 min. Then quantify the gel to calculate the % yield.

File:ZhangLab 2 2008-04-01 18hr 26min-.jpeg

Result: The right most lane is the 100 bp ladder, which has a lot of smear in the figure above. This is due to the reason that the concentration is too high compare to the amount of ladder added into the buffer. Instead, only 0.5 - 1 uL of ladder is needed to dilute with water. That way will greatly reduce the smear. Two and Threelanes to the right are the reference DNA 90-mer, which is used for quantification purpose. Four and Five lanes to the right are the sample from 27k padlock library from last experiment (the one with two bright bands on 2D gel, and this is the bottom band). The bands seems to have close intensity with the 90-mer bands, therefore I have enough concentration for capture. After quantification, I determined that the probe has 6.5 ng /uL.

Experiment 2: Capturing genomic targets with padlock probes

 1.Circularization Reaction:
 Jurkat genomic DNA (100 ng/ uL):    2 uL
 CE27K probe                    :    2 uL
 10X ampLigase buffer           :    1 uL
 H2O                            :    5 uL