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Daniel:Notebook/HiResChrPaint/2013-4-27
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=Probe Prep Test ([[Daniel:Notebook/HiResChrPaint/2013-4-22|Started 4/22/2013]])= [[Daniel:Notebook/HiResChrPaint|Back to calendar]] ==Ethanol Precipitation (Continued from [[Daniel:Notebook/HiResChrPaint/2013-4-26|4/26/2013]])== 5. Took samples from -80 and centrifuged at 14000 rpm for 25 minutes at 4 C 6. Removed supernatant and added 750 uL 75% EtOH 7. Centrifugd at 14000 rpm for 15 minutes at 4 C 8. Removed most supernatant with pipette and then dried in hood 9. Resuspended in 8 uL nuclease free water ==TBU gel Quantification== 1. Preheated TBE buffer in 60 C incubator 2. microwaved for 1 minute 3. Added hot TBE to gel box and immediately preran gel for 20 minutes at 250V 4. Created samples according to following table {| 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="15" | Lane | align="center" width="65" | 3 | align="center" width="65" | 4 | align="center" width="65" | 5 | align="center" width="65" | 6 | align="center" width="65" | 7 | align="center" width="65" | 8 | align="center" width="65" | 9 | align="center" width="65" | 10 |- style="font-size:12pt" align="center" | height="15" | Sample | Ladder | Ladder | V4S3 | V4S3 | V6S3 | V6S3 | Ladder | Buffer |- 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 |- style="font-size:12pt" align="center" | height="15" | uL hot TBE | align="center" | 5.5 | align="center" | 5 | align="center" | 5.5 | align="center" | 5.5 | align="center" | 5.5 | align="center" | 5.5 | align="center" | 5.5 | align="center" | 6 |- style="font-size:12pt" align="center" | height="15" | uL 2x buffer | align="center" | 6 | align="center" | 6 | align="center" | 6 | align="center" | 6 | align="center" | 6 | align="center" | 6 | align="center" | 6 | align="center" | 6 |- style="font-size:12pt" align="center" | height="15" | loaded | align="center" | 12 | align="center" | 12 | align="center" | 12 | align="center" | 12 | align="center" | 12 | align="center" | 12 | align="center" | 12 | align="center" | 12 |} 5. Incubated samples at 65C for 8 minutes 6. Cold snapped with ice box 7. Loaded 12 uL each sample and ran gel for 20 minutes at 250V 8. Stained with 3 uL SYBR gold for 25 minutes ===Gel Results=== Volume Report: [[File:GelQuant_042713.xls]] [[Image:GelQuant_042713.jpg|600px]] [[Image:GelQuant_analysis_042713.jpg|600px]] ===Sample Concentrations=== {| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext> |- align="center" valign="bottom" | width="69" height="12" | Sample | width="69" | ng/uL | width="69" | ng (7 uL) |- align="center" valign="bottom" | height="12" | V4S3 | align="center" | 4.6 | align="center" | 32.4 |- align="center" valign="bottom" | height="12" | V4S3 | align="center" | 3.1 | align="center" | 22.0 |- align="center" valign="bottom" | height="12" | V6S3 | align="center" | 3.5 | align="center" | 24.7 |} ==Discussion== With the abysmally low yields from the Lambda Exonuclease reaction, I have come to the conclusions that it is this reaction causing the most problems in my probe prep protocol. I'm going to need to approach this reaction better before I continue. The following table summarizes the yields in (ug and %) comparing my probe preps and Noi's: {| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext> |- style="font-size:12pt" align="center" | align="center" width="65" height="42" | | width="65" | qPCR (ug) | width="50" | qPCR Yield (%) | width="65" | DpnII (ug) | width="71" | DpnII Yield (%) | width="65" | Lambda Exo (ug) | width="65" | Lambda Exo Yield (%) | width="65" | Yield from DPNII |- style="font-size:12pt" align="center" valign="bottom" |style="font-weight:bold" height="15" | Me | align="center" | | align="center" | | align="center" | | align="center" | | align="center" | | align="center" | | align="center" | |- style="font-size:12pt" align="center" valign="bottom" | height="15" | V4S3 | align="center" | 8.2 | align="center" | 100 | align="center" | 5.6 | align="center" | 68.3 | align="center" | 0.03 | align="center" | 0.4 | align="center" | 0.6 |- style="font-size:12pt" align="center" valign="bottom" | height="15" | V6S3 | align="center" | 7.4 | align="center" | 100 | align="center" | 4.7 | align="center" | 63.5 | align="center" | 0.02 | align="center" | 0.3 | align="center" | 0.5 |- style="font-size:12pt" align="center" valign="bottom" |style="font-weight:bold" height="15" | Noi | align="center" | | align="center" | | align="center" | | align="center" | | align="center" | | align="center" | | align="center" | |- style="font-size:12pt" align="center" valign="bottom" | height="15" | V4S3 | align="center" | 13.2 | align="center" | 100 | align="center" | 8.9 | align="center" | 67.2 | align="center" | 2.2 | align="center" | 16.4 | align="center" | 24.4 |- style="font-size:12pt" align="center" valign="bottom" | height="15" | V6S3 | align="center" | 15.9 | align="center" | 100 | align="center" | 12.5 | align="center" | 78.4 | align="center" | 2.6 | align="center" | 16.1 | align="center" | 20.5 |} [[Daniel:Notebook/HiResChrPaint|Back to calendar]]
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