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=Probe Prep Test ([[Daniel:Notebook/HiResChrPaint/2013-4-22|Started 4/22/2013]])= [[Daniel:Notebook/HiResChrPaint|Back to calendar]] ==TBU Gel Quantification== Purpose: Test quantification results from [[Daniel:Notebook/HiResChrPaint/2013-4-29|samples]] digested with lambda exonuclease at different time/concentrations. ===Protocol=== 1. Set up gel according to following table; following [[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" align="center" |style="font-weight:bold" width="65" height="15" | Lane | align="center" width="65" | 2 | 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 |- style="font-size:12pt" align="center" |style="font-weight:bold" height="53" | Sample | 0.5 uL Low Mass ladder | 1 uL LM ladder | Control (10x exo, 1 hr) | 1X lambda exo, 1 hr | 10 x lambda exo, 30 min | 10 x lambda exo, 2 hr | V4S3 | V6S3 |- style="font-size:12pt" | height="15" valign="bottom" | Probe Set | valign="bottom" | NA | valign="bottom" | NA | valign="bottom" | V6S3 | valign="bottom" | V6S2 | valign="bottom" | V6S4 | valign="bottom" | V4S2 | [[Daniel:Notebook/HiResChrPaint/2013-4-27|V4S3]] | [[Daniel:Notebook/HiResChrPaint/2013-4-27|V6S3]] |} ===Results=== Volume Report: [[File:TBUGel_050113.xls]] [[Image:TBUGel_050113.jpg|600px]] [[Image:TBUGel_050113_analysis.jpg|600px]] ===Quantitation=== {| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext> |- style="font-weight:bold" align="center" valign="bottom" | width="69" height="12" | Sample | width="69" | ng on gel | width="69" | ng/uL | width="69" | ng in 6 uL |- align="center" valign="bottom" | height="12" | V4S3 | align="center" | 11.5 | align="center" | 23.0 | align="center" | 137.9 |- align="center" valign="bottom" | height="12" | V6S3 | align="center" | 10.9 | align="center" | 21.9 | align="center" | 131.2 |} ===Discussion=== It would appear that too little DNA was left to quantify once everything was said and done. Not surprising, since all of these reactions started with very little DNA. The V4S3 and V6S3 (from [[Daniel:Notebook/HiResChrPaint/2013-4-27|4/27/2013]]) samples I was able to classify. Today's quantification showed around 10 times the amount of DNA present as the 4/27 gel. It would appear that this gel quantification is quite erroneous in many ways. Not sure how Noi got such great results. Looking at the gel qualitatively, I would say that the 2 hour digestion has almost no product left. That being said, the 30 minute digest should have more left than the 1 hour, but we do not see this response. Another thing to remember is that all of these are different probe sets, and may have very different behaviors. In short, I don't know that I can draw any definite conclusions from this gel, other than the lambda exonuclease reaction, in general, is causing me to lose too much DNA. ==TBE Gel== Purpose: I want to rerun the lambda digestion experiment (above) but using the same set of DNA. I also want to keep a batch immediately after digestion to make sure I'm not losing too much DNA in the ethanol precipitation. I have identified two possible samples that have around 1 ug each, but I am unsure of which stage they are at. This TBE gel should tell me. ===Protocol=== 1. Loaded gel according to 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" valign="bottom" | width="65" height="15" | Lane | align="center" width="65" | 3 | align="center" width="65" | 4 | align="center" width="65" | 5 |- style="font-size:12pt" align="center" valign="bottom" |style="font-weight:bold" height="15" | Sample | 25 bp ladder | V4S2 | V4S3 |- style="font-size:12pt" align="center" valign="bottom" |style="font-weight:bold" height="15" | uL sample | align="center" | 0.5 | align="center" | 0.5 | align="center" | 0.5 |- style="font-size:12pt" align="center" valign="bottom" |style="font-weight:bold" height="15" | uL TBE | align="center" | 5.5 | align="center" | 5.5 | align="center" | 5.5 |- style="font-size:12pt" align="center" valign="bottom" |style="font-weight:bold" height="15" | uL 6x dye | align="center" | 2 | align="center" | 2 | align="center" | 2 |- style="font-size:12pt" align="center" valign="bottom" |style="font-weight:bold" height="15" | uL | align="center" | 7 | align="center" | 7 | align="center" | 7 |} 2. Ran gel for 25 minutes at 200V 3. Stained with 3 uL SYBR gold for 2 minutes ===Results=== [[Image:TBEgel_050113.jpg|600px]] Clearly, these are pre-DpnII digestion. Proceeding to DpnII digest. ==DpnII Digestion== 1. Set up reaction according to table *DpnII-Lot 0091201; Buffer 0031105 {| 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="32" | Sample | width="65" | ng/uL | width="65" | uL | width="65" | ng | width="65" | uL 10x buffer | width="65" | uL DpnII (50 U/uL) | width="65" | uL nf H20 | width="65" | Total |- style="font-size:12pt" align="center" | height="15" | V4S2 | align="center" | 139.7 | align="center" | 10 | align="center" | 1397 | align="center" | 5 | align="center" | 1 | align="center" | 34 | align="center" | 50 |- style="font-size:12pt" align="center" | height="15" | V6S2 | align="center" | 111.9 | align="center" | 10 | align="center" | 1119 | align="center" | 5 | align="center" | 1 | align="center" | 34 | align="center" | 50 |} 2. Incubate 16 hours at 37 C followed by 20 minutes at 65 C for heat shock (continued on [[Daniel:Notebook/HiResChrPaint/2013-5-2|5/2/2013]]) [[Daniel:Notebook/HiResChrPaint|Back to calendar]]
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