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Daniel:Notebook/HiResChrPaint/2013-6-21
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=S1 Probe Preparation (Started [[Daniel:Notebook/HiResChrPaint/2013-6-20|6/20/2013]])= [[Daniel:Notebook/HiResChrPaint|Back to Calendar]] ==DpnII Digestion (Continued from [[Daniel:Notebook/HiResChrPaint/2013-6-20|6/20/2013]])== 3. Pooled all V4 samples (4->1) and 2 V6 samples (4->2) 4. [[Daniel:Protocols/Qiaquick_Column|Column purification]], elute with 40 uL EB 5. Measure Concentration in Nanodrop 6. TBE gel to ensure proper digestion ==Nanodrop Results== {| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext> |- style="font-size:12pt;font-weight:bold" align="center" | width="96" height="15" | Sample | width="65" | V4S1 | width="65" | V6S1AB | width="65" | V6S1CD |- style="font-size:12pt" align="center" |style="font-weight:bold" height="15" | ng/uL | align="center" | 66.7 | align="center" | 118.3 | align="center" | 120.6 |- style="font-size:12pt" align="center" |style="font-weight:bold" height="15" | ug in 40 uL | align="center" | 2.7 | align="center" | 4.7 | align="center" | 4.8 |} ===TBE gel=== 1. Prepared samples 5.5 uL TBE, 0.5 uL sample, 2 uL 6x dye 2. Sample matrix {| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext> |- style="font-size:12pt;font-weight:bold" align="center" | width="65" height="15" | Lane | 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 |- style="font-size:12pt" align="center" |style="font-weight:bold" height="30" | Sample | 25 bp ladder | V4S1B Undigested | V6S1B Undigested | V4S1 | V6S1AB | V6S1CD |} 3. Ran gel for 20 minutes at 220 V, stained for 2 minutes with 3 uL SYBR gold ===Gel Image=== [[Image:DpnIIDigest_062113.jpg|600px]] ==Lambda Exonuclease digestion== 1. Reaction table {| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext> |- style="font-size:12pt;font-weight:bold" align="center" | width="96" height="15" | Sample | width="65" | V4S1 | width="65" | V6S1AB | width="65" | V6S1CD |- style="font-size:12pt" align="center" |style="font-weight:bold" height="15" | ng/uL | align="center" | 66.7 | align="center" | 118.3 | align="center" | 120.6 |- style="font-size:12pt" align="center" |style="font-weight:bold" height="15" | ug in 40 uL | align="center" | 2.7 | align="center" | 4.7 | align="center" | 4.8 |- style="font-size:12pt" align="center" |style="font-weight:bold" height="15" | uL 10x buffer | align="center" | 5 | align="center" | 5 | align="center" | 5 |- style="font-size:12pt" align="center" |style="font-weight:bold" height="30" | uL Lambda Exonuclease | align="center" | 3 | align="center" | 5 | align="center" | 5 |- style="font-size:12pt" align="center" |style="font-weight:bold" height="15" | uL nfH2O | align="center" | 7 | align="center" | 5 | align="center" | 5 |- style="font-size:12pt" align="center" |style="font-weight:bold" height="15" | total | align="center" | 50 | align="center" | 50 | align="center" | 50 |} 3. Incubate at 37C for 1 hour followed by 10 minute heat shock at 75C 4. ssDNA column purification, elute with 10 uL ==TBU gel== 1. Sample Matrix; followed [[Daniel:Protocols/TBUgel|Protocol 2]] {| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext> |- style="font-size:12pt;font-weight:bold" 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 |- style="font-size:12pt" align="center" |style="font-weight:bold" height="30" | Sample | 0.5 uL LM ladder | 1 uL LM ladder | V4 (1:10) | V6 (1:10) | V4 (1:20) | V6 (1:20) |- style="font-size:12pt" align="center" |style="font-weight:bold" height="15" | uL sample | align="center" | 0.5 | align="center" | 1 | align="center" | 0.5 | align="center" | 0.5 | 2* | 2** |- style="font-size:12pt" align="center" |style="font-weight:bold" height="15" | uL TBE | align="center" | 1.5 | align="center" | 1 | align="center" | 9.5 | align="center" | 9.5 | align="center" | 1 | align="center" | 1 |- style="font-size:12pt" align="center" |style="font-weight:bold" height="30" | uL 2X Urea Buffer | align="center" | 2 | align="center" | 2 | align="center" | 10 | align="center" | 10 | align="center" | 1 | align="center" | 1 |- style="font-size:12pt" align="center" |style="font-weight:bold" height="15" | Loaded | align="center" | 4 | align="center" | 4 | align="center" | 4 | align="center" | 4 | align="center" | 4 | align="center" | 4 |} Note: *denotes sample taken from lane 5 sample; **denotes sample taken from lane 6 sample 2. Ran gel for 15 minutes at 250V 3. Stained with 3 uL SYBR gold for 10 minutes ===TBU gel Results=== Volume Report: [[File:GelQuant_062113.xls]] [[Image:GelQuant062113.jpg|600px]] [[Image:GelQuant062113_Volume.jpg|600px]] {| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext> |- style="font-weight:bold" align="center" | width="69" height="12" | Sample | width="69" | V4S1 | width="69" | V6S1 |- align="center" |style="font-weight:bold" height="12" | ~ng/uL | align="center" | 60 | align="center" | 140 |- align="center" |style="font-weight:bold" height="12" | ug in 10 uL | align="center" | 0.6 | align="center" | 1.4 |}
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