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Alice:LabNotes/2007-11-16
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PCR purification (QIAquick) on post-PCR product (reaction with newly designed primer rs1264899)<br> <b>Protocol:</b> <br> Add 5 volumes of Buffer PBI to 1 volume of PCR sample and mix.<br> Place a QIAquick spin column in a provided 2 ml collection tube.<br> To bind DNA, apply the sample to the QIAquick column and centrifuge for 1 min.<br> Discard flow-through, and place the column back into the tube.<br> Add 0.75 ml PE buffer to QIAquick column and centrifuge for 1 min.<br> Discard the flow-through and spin for additional min.<br> Place the column in a clean 1.5 ml microcentrifuge tube.<br> Add 30 ul EB buffer to center of the QIAquick membrane and centrifuge the column for 1 min to elute DNA. <br> <b> Note: Store the yielded product in -20 freezer, otherwise the sample will evaporate in -4 fridge.</b> <br> Then I ran a gel on the yielded products. Each well contain total of 5 ul of mixture of reagents.<br> <b>Summary of volumes of Reagents used:</b> {| {{table}} | align="center" style="background:#f0f0f0;"|'''''' | align="center" style="background:#f0f0f0;"|'''well 1-2''' | align="center" style="background:#f0f0f0;"|'''well 3-4''' | align="center" style="background:#f0f0f0;"|'''well 5-8''' |- | Low Mass bp Ladder ||0 ul ||3 ul ||0 ul|| |- | 100 bp Ladder ||3 ul ||0 ul ||0 ul|| |- | TBE buffer || 1 ul|| 1 ul|| 0 ul|| |- | Novex 5x TBE loading dye || 1 ul|| 1 ul|| 1 ul|| |- | post-PCR sample || 0 ul|| 0 ul|| 4 ul|| |- | |} <b>Results:</b><br> [[Image:ZhangLab_2 2007-11-16-11hr 07min.png]] <br> First of all, the intensity of the gel was not bright enough. This indicates that either the gel didn't get stained long enough or the staining solution has gone bad. But the second condition was very unlikely since professor Zhang ran some very good gel with the same staining solution. Also, the focus of the gel imaging machine could be adjusted to be either further away of closer to the level of samples, since the image shows some fuzzy edges.
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