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=Probe Prep Test ([[Daniel:Notebook/HiResChrPaint/2013-4-22|Started 4/22/2013]])= [[Daniel:Notebook/HiResChrPaint|Back to Calendar]] ==Column Purification vs. Ethanol Precipitation== ===Ethanol Precipitation (continued from [[Daniel:Notebook/HiResChrPaint/2013-5-6|5/6/2013]])=== 5. Centrifuged 30 min at 10000 rpm at 4C 6. Removed supernatant and added 750 uL chilled EtOH 7. Centrifuged 15 min at 10000 rpm at 4C 8. Removed supernatant and dried in hood 9. Resuspended in 10 uL nf H20 ===TBU Gel comparison=== [[Daniel:Protocols/TBUgel|Protocol 2]] 1. Gel lanes {| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext> |- style="font-size:12pt" align="center" | width="65" height="39" | Sample | width="50" | 0.5 Ladder | width="50" | 1 uL Ladder | width="50" | Original | width="50" | Column (1uL) | width="50" | Column (2uL) | width="50" | EtOH (1 uL) A | width="50" | EtOH (2uL) A | width="50" | EtOH (1 uL) B | width="50" | EtOH (2uL) B |- style="font-size:12pt" align="center" | height="15" | uL sample | align="center" | 0.5 | align="center" | 1 | align="center" | 0.5 | align="center" | 0.5 | align="center" | 0.5 | align="center" | 0.5 | align="center" | 0.5 | align="center" | 0.5 | align="center" | 0.5 |- style="font-size:12pt" align="center" | height="15" | uL TBE | align="center" | 9.5 | align="center" | 9 | align="center" | 9.5 | align="center" | 9.5 | align="center" | 9.5 | align="center" | 9.5 | align="center" | 9.5 | align="center" | 9.5 | align="center" | 9.5 |- style="font-size:12pt" align="center" | height="15" | uL 2X buffer | align="center" | 10 | align="center" | 10 | align="center" | 10 | align="center" | 10 | align="center" | 10 | align="center" | 10 | align="center" | 10 | align="center" | 10 | align="center" | 10 |- style="font-size:12pt" align="center" | height="15" | Loaded | align="center" | 4 | align="center" | 4 | align="center" | 4 | align="center" | 4 | align="center" | 4 | align="center" | 4 | align="center" | 4 | align="center" | 4 | align="center" | 4 |- style="font-size:12pt" align="center" | height="15" | Dilution | align="center" | 0.1 | align="center" | 0.1 | align="center" | 0.1 | align="center" | 0.1 | align="center" | 0.1 | align="center" | 0.1 | align="center" | 0.1 | align="center" | 0.1 | align="center" | 0.1 |} 2. Heat shock 10 minutes at 75 C 3. Run gel for 24 minutes at 250V 4. Stain with 3 uL SYBR gold for 20 minutes ===Gel Results=== Volume Report: [[File:PrimerQuant_050813.xls]] [[Image:PrimerQuant_050813.jpg|600px]] [[Image:PrimerQuant_050813_Volume.jpg|600px]] ===Quantification Results=== {| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext> |- align="center" valign="bottom" | align="center" width="69" height="12" | | width="69" | ng/uL | width="69" | Yield | width="69" | Fold Increase (2uL/1uL) |- align="center" valign="bottom" | height="12" | Original | align="center" | 1926.4 | align="center" | 139.8 | align="center" | |- align="center" valign="bottom" | height="12" | 1 uL column | align="center" | 906.6 | align="center" | 65.8 | align="center" | |- align="center" valign="bottom" | height="12" | 2 uL column | align="center" | 1337.0 | align="center" | 97.0 | align="center" | 1.5 |- align="center" valign="bottom" | height="12" | 1 uL EtOH A | align="center" | 205.5 | align="center" | 14.9 | align="center" | |- align="center" valign="bottom" | height="12" | 2 uL EtOH A | align="center" | 676.0 | align="center" | 49.1 | align="center" | 3.3 |- align="center" valign="bottom" | height="12" | 1 uL EtOH B | align="center" | 204.6 | align="center" | 14.8 | align="center" | |- align="center" valign="bottom" | height="12" | 2 uL EtOH B | align="center" | 549.2 | align="center" | 39.9 | align="center" | 2.7 |} ===Discussion=== Looks like the column purification is better than Ethanol precipitation once we get this low. As such, I'm going to start my next probe set with more DNA and then when I get to the lambda digestion I'll purify with the column. I also think that the amount of starting material for the ethanol precipitation affects the yield. I think that starting with more DNA will give a higher % yield, because some DNA will always be left in solution. This would be confirmed if the gel quantification results in less than 1/2 yields in the 1uL samples compared with the 2.
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