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=Padlock Probes ([[Daniel:Notebook/HiResChrPaint/2014-8-13|Started 8/13/2014]])= [[ Daniel:Notebook/HiResChrPaint|Back to Calendar]] ==Lambda Exonuclease Digestion== Since it looks like I might have less DNA than I originally thought (makes sense, several hundred ug is pretty unlikely), I'm going to run the lambda exonuclease digestion on the remaining padlock probes, A and B. '''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" |style="background-color:#C5D9F1" width="151" height="30" | Reagent |style="background-color:#FCD5B4" width="65" | PPRL-A (uL) |style="background-color:#FCD5B4" width="65" | PPRL-B (uL) |- style="font-size:12pt" |style="background-color:#C5D9F1" height="15" valign="bottom" | Amplicon | align="center" align="center" valign="bottom" | 45 | align="center" align="center" valign="bottom" | 55 |- style="font-size:12pt" |style="background-color:#C5D9F1" height="15" valign="bottom" | ug Input | align="center" align="center" valign="bottom" | 20.7 | align="center" align="center" valign="bottom" | 25.6 |- style="font-size:12pt" |style="background-color:#C5D9F1" height="15" valign="bottom" | Lambda Exo Buffer (10X) | align="center" align="center" valign="bottom" | 10 | align="center" align="center" valign="bottom" | 10 |- style="font-size:12pt" |style="background-color:#C5D9F1" height="15" valign="bottom" | Lambda Exonuclease (5 U/uL) | align="center" align="center" valign="bottom" | 5 | 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" | 40 | align="center" align="center" valign="bottom" | 30 |- style="font-size:12pt" |style="background-color:#C5D9F1" height="15" valign="bottom" | Total |style="font-weight:bold" align="center" align="center" valign="bottom" | 100 |style="font-weight:bold" align="center" align="center" valign="bottom" | 100 |} #Set up reaction according to table #Incubate at 37C for '''2 hours''' #Heat inactivate for 10 minutes at 75C #Purify using [[Daniel:Protocols/ssDNA Column|ssDNA columns]], elute with 20 uL nfH2O ===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" | width="65" height="31" | Sample | width="65" | ng/uL | width="65" | ug in 20 uL | width="65" | Input (ug) | width="77" | Efficiency (%) | width="65" | ug Total |- style="font-size:12pt" |style="background-color:#D9D9D9" height="15" valign="bottom" | PPRL-A | align="center" align="center" valign="bottom" | 132.8 | align="center" align="center" valign="bottom" | 2.7 | align="center" align="center" valign="bottom" | 20.7 | align="center" align="center" valign="bottom" | 12.83 | 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" | 214 | align="center" align="center" valign="bottom" | 4.3 | align="center" align="center" valign="bottom" | 25.6 | align="center" align="center" valign="bottom" | 16.72 |style="font-weight:bold" align="center" align="center" valign="bottom" | 6.9 |} This result is good enough to proceed with. ==USER/DpnII Digestion== '''Reaction Table''' {| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext> |- style="background-color:#B8CCE4;font-size:12pt;font-weight:bold" align="center" | width="93" height="33" | Reagent | width="65" | PPRL (uL) |- style="font-size:12pt" |style="background-color:#FCD5B4" height="15" valign="bottom" | amplicon | align="center" align="center" valign="bottom" | 30 |- style="font-size:12pt" |style="background-color:#FCD5B4" height="15" valign="bottom" | ug Input | align="center" align="center" valign="bottom" | 2.0 |- style="font-size:12pt" |style="background-color:#FCD5B4" height="15" valign="bottom" | USER | align="center" align="center" valign="bottom" | 5 |- style="font-size:12pt" |style="background-color:#FCD5B4" height="15" valign="bottom" | nf H2O | align="center" align="center" valign="bottom" | 45 |- style="font-size:12pt;font-weight:bold" |style="background-color:#FCD5B4" height="15" valign="bottom" | Total | align="center" align="center" valign="bottom" | 80 |} #Set up reaction according to table in a 0.2 mL PCR tube #Incubate for 2 hours at 37C #Add 15 uL according to DpnII Primer table (below) '''DpnII Primer Table''' {| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext> |- style="background-color:#B8CCE4;font-size:12pt;font-weight:bold" align="center" | width="93" height="31" | Reagent | width="65" | PPRL (uL) |- style="font-size:12pt" |style="background-color:#FCD5B4" height="15" valign="bottom" | 10X DpnII Buffer | align="center" align="center" valign="bottom" | 10 |- style="font-size:12pt" |style="background-color:#FCD5B4" height="30" valign="bottom" | 100 uM RE-DpnII-V4 guide oligo | align="center" align="center" valign="bottom" | 5 |- style="font-size:12pt;font-weight:bold" |style="background-color:#FCD5B4" height="15" valign="bottom" | Total | align="center" align="center" valign="bottom" | 15 |} #Incubate at 94C for 2 minutes #Cool to 37C and incubate for 3 minutes #Add 5 uL DpnII and incubate at 37C for 2 hours #Heat inactivate DpnII at 65C for 20 minutes ===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" | width="86" height="28" valign="bottom" | Sample | width="65" align="center" | ng/uL ssDNA | width="65" align="center" | ug in 20 uL | width="65" align="center" | Total |- style="font-size:12pt" |style="background-color:#D9D9D9" height="15" valign="bottom" | PPRL-DpnII-A | align="center" align="center" valign="bottom" | 38.7 | align="center" align="center" valign="bottom" | 0.8 | align="center" align="center" valign="bottom" | |- style="font-size:12pt" |style="background-color:#D9D9D9" height="15" valign="bottom" | PPRL-DpnII-B | align="center" align="center" valign="bottom" | 35.3 | align="center" align="center" valign="bottom" | 0.7 |style="font-weight:bold" align="center" align="center" valign="bottom" | 1.5 |} Should be enough to label with. [[Category:HRCP]] [[Category:Lambda Exo]] [[Category:DpnII]] [[Category:USER]] [[Category:20140813]]
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