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=Padlock Probes ([[Daniel:Notebook/HiResChrPaint/2014-8-13|Started 8/13/2014]])= [[ Daniel:Notebook/HiResChrPaint|Back to Calendar]] ==Ethanol Precipitation ([[Daniel:Notebook/HiResChrPaint/2014-8-18|From 8/18/2014]])== #Remove from -80C centrifuge at 4400 rpm for 30 minutes at 4C #Add 700 uL 75% EtOH ##Break up the pellet and transfer to a 1.5mL tube #Centrifuge once more at 14000 rpm for 10 minutes #Take out supernatant and dry in laminar flow hood for 20 minutes #Resuspend in 20 uL nf H2O ===Nanodrop Results=== {| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext> |- style="background-color:#CCC0DA;font-size:12pt" align="center" valign="bottom" | width="65" height="15" | Sample | width="65" | ng/uL (1:5) | width="65" | ng/uL | width="65" | ug in 60 uL | width="65" | ug total |- style="font-size:12pt" |style="background-color:#D9D9D9" height="15" valign="bottom" | PPRL-A | align="center" align="center" valign="bottom" | 459.9 | align="center" align="center" valign="bottom" | 2,299.5 | align="center" align="center" valign="bottom" | 138.0 | align="center" align="center" valign="bottom" | |- style="font-size:12pt" |style="background-color:#D9D9D9" height="15" valign="bottom" | PPRL-B | align="center" align="center" valign="bottom" | 465.9 | align="center" align="center" valign="bottom" | 2,329.5 | align="center" align="center" valign="bottom" | 139.8 |style="font-weight:bold" align="center" align="center" valign="bottom" | 277.7 |} This seems to be a pretty ridiculous amount of probe, but it is consistent with my original nanodrop reading (4822.2 ng/uL of a 20 uL sample, undiluted). I'll push ahead cautiously with a large amount of the probe (but not all). ==Lambda Exonuclease digestion== I'll just be using 10 uL of PPRL-A for this one, since it would appear that 10 uL is approximate 20 ug of probe. '''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" |style="background-color:#C5D9F1" width="112" height="15" valign="bottom" | Reagent |style="background-color:#FCD5B4" width="65" align="center" valign="bottom" | uL |- style="font-size:12pt" |style="background-color:#C5D9F1" height="15" valign="bottom" | Amplicon | align="center" align="center" valign="bottom" | 10 |- style="font-size:12pt" |style="background-color:#C5D9F1" height="15" valign="bottom" | ug in 10 uL | align="center" align="center" valign="bottom" | 23.0 |- style="font-size:12pt" |style="background-color:#C5D9F1" height="30" valign="bottom" | Lambda Exonuclease Buffer (10X) | align="center" align="center" valign="bottom" | 10 |- style="font-size:12pt" |style="background-color:#C5D9F1" height="30" valign="bottom" | Lambda Exonuclease (5 U/uL) | align="center" align="center" valign="bottom" | 5 |- style="font-size:12pt" |style="background-color:#C5D9F1" height="15" valign="bottom" | nf H2O | align="center" align="center" valign="bottom" | 75 |- style="font-size:12pt" |style="background-color:#C5D9F1" height="15" valign="bottom" | Total |style="font-weight:bold" align="center" align="center" valign="bottom" | 100 |} #Set up reaction according to table #Incubate at 37C for 1 hour #Heat inactivate for 10 minutes at 75C #Purify using 2 [[Daniel:Protocols/ssDNA Column|ssDNA columns]], elute with 20 uL nfH2O each ===Nanodrop Results=== Sample A reported 2.1 ng/uL and sample B reported 1.7 ng/uL. This means just about everything was lost. ==Gel Quantification Check== Based on the results above (no sample after Lambda digestion), I'm going to do a massive gel dilution series to see approximately what the concentration of PPRL-A is. I'll either repeat for B, or more likely assume it is on the same scale. {| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext> |- style="background-color:#E6B8B7;font-size:12pt;font-weight:bold" align="center" | width="86" height="34" | Lane | width="86" | 2 (Ladder) | 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" |style="background-color:#C5D9F1" height="15" valign="bottom" | Dilution | align="center" align="center" valign="bottom" | 1 | align="center" align="center" valign="bottom" | 1 | align="center" align="center" valign="bottom" | 1 | align="center" align="center" valign="bottom" | 10 | align="center" align="center" valign="bottom" | 10 | align="center" align="center" valign="bottom" | 10 | align="center" align="center" valign="bottom" | 10 | align="center" align="center" valign="bottom" | 50 | align="center" align="center" valign="bottom" | 50 |- style="font-size:12pt" |style="background-color:#C5D9F1" height="15" valign="bottom" | uL Sample |style="background-color:#D9D9D9" align="center" align="center" valign="bottom" | 1 |style="background-color:#D9D9D9" align="center" align="center" valign="bottom" | 1 |style="background-color:#D9D9D9" align="center" align="center" valign="bottom" | 0.5 |style="background-color:#D9D9D9" align="center" align="center" valign="bottom" | 4 |style="background-color:#D9D9D9" align="center" align="center" valign="bottom" | 2 |style="background-color:#D9D9D9" align="center" align="center" valign="bottom" | 1 |style="background-color:#D9D9D9" align="center" align="center" valign="bottom" | 0.5 |style="background-color:#D9D9D9" align="center" align="center" valign="bottom" | 1 |style="background-color:#D9D9D9" align="center" align="center" valign="bottom" | 0.5 |- style="font-size:12pt" |style="background-color:#C5D9F1" height="15" valign="bottom" | uL TBE | align="center" align="center" valign="bottom" | 4.5 | align="center" align="center" valign="bottom" | 4.5 | align="center" align="center" valign="bottom" | 5 | align="center" align="center" valign="bottom" | 1.5 | align="center" align="center" valign="bottom" | 3.5 | align="center" align="center" valign="bottom" | 4.5 | align="center" align="center" valign="bottom" | 5 | align="center" align="center" valign="bottom" | 4.5 | align="center" align="center" valign="bottom" | 5 |- style="font-size:12pt" |style="background-color:#C5D9F1" height="15" valign="bottom" | uL buffer |style="background-color:#D9D9D9" align="center" align="center" valign="bottom" | 2 |style="background-color:#D9D9D9" align="center" align="center" valign="bottom" | 2 |style="background-color:#D9D9D9" align="center" align="center" valign="bottom" | 2 |style="background-color:#D9D9D9" align="center" align="center" valign="bottom" | 2 |style="background-color:#D9D9D9" align="center" align="center" valign="bottom" | 2 |style="background-color:#D9D9D9" align="center" align="center" valign="bottom" | 2 |style="background-color:#D9D9D9" align="center" align="center" valign="bottom" | 2 |style="background-color:#D9D9D9" align="center" align="center" valign="bottom" | 2 |style="background-color:#D9D9D9" align="center" align="center" valign="bottom" | 2 |- style="font-size:12pt" |style="background-color:#C5D9F1" height="15" valign="bottom" | Total | align="center" align="center" valign="bottom" | 7.5 | align="center" align="center" valign="bottom" | 7.5 | align="center" align="center" valign="bottom" | 7.5 | align="center" align="center" valign="bottom" | 7.5 | align="center" align="center" valign="bottom" | 7.5 | align="center" align="center" valign="bottom" | 7.5 | align="center" align="center" valign="bottom" | 7.5 | align="center" align="center" valign="bottom" | 7.5 | align="center" align="center" valign="bottom" | 7.5 |- style="font-size:12pt" |style="background-color:#C5D9F1" height="15" valign="bottom" | uL Added |style="background-color:#D9D9D9" align="center" align="center" valign="bottom" | 7.2 |style="background-color:#D9D9D9" align="center" align="center" valign="bottom" | 7.2 |style="background-color:#D9D9D9" align="center" align="center" valign="bottom" | 7.2 |style="background-color:#D9D9D9" align="center" align="center" valign="bottom" | 7.2 |style="background-color:#D9D9D9" align="center" align="center" valign="bottom" | 7.2 |style="background-color:#D9D9D9" align="center" align="center" valign="bottom" | 7.2 |style="background-color:#D9D9D9" align="center" align="center" valign="bottom" | 7.2 |style="background-color:#D9D9D9" align="center" align="center" valign="bottom" | 7.2 |style="background-color:#D9D9D9" align="center" align="center" valign="bottom" | 7.2 |- style="font-size:12pt;font-weight:bold" |style="background-color:#C5D9F1" height="15" valign="bottom" | Final uL Added | align="center" align="center" valign="bottom" | 0.96 | align="center" align="center" valign="bottom" | 0.96 | align="center" align="center" valign="bottom" | 0.48 | align="center" align="center" valign="bottom" | 0.384 | align="center" align="center" valign="bottom" | 0.192 | align="center" align="center" valign="bottom" | 0.096 | align="center" align="center" valign="bottom" | 0.048 | align="center" align="center" valign="bottom" | 0.0192 | align="center" align="center" valign="bottom" | 0.0096 |} #Run for 30 minutes at 200 V '''Gel Image''' [[Image:2014-08-19-PPRL-A.jpg|500px]] [[Category:HRCP]] [[Category:Lambda Exo]] [[Category:TBE Gel]] [[Category:20140813]]
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