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=Strand Control ([[Daniel:Notebook/HiResChrPaint/2014-7-8|Started 07/08/2014]])= [[Daniel:Notebook/HiResChrPaint|Back to Calendar]] ==DpnII Digest ([[Daniel:Notebook/HiResChrPaint/2014-7-8#DpnII Digestion|from 07/08/2014]])== #[[Daniel:Protocols/Qiaquick Column|Column Purification]], elute with 40 uL #Measure concentration in nanodrop ===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" | width="65" height="15" valign="bottom" | Sample | width="65" align="center" valign="bottom" | V6S2AB | width="65" align="center" valign="bottom" | V6S2CD | width="65" align="center" valign="bottom" | V6S3AB | width="65" align="center" valign="bottom" | V6S3CD |- style="font-size:12pt" |style="font-weight:bold" height="15" valign="bottom" | ng/uL | align="center" align="center" valign="bottom" | 122.4 | align="center" align="center" valign="bottom" | 110.7 | align="center" align="center" valign="bottom" | 137 | align="center" align="center" valign="bottom" | 133.5 |- style="font-size:12pt" |style="font-weight:bold" height="15" valign="bottom" | ug in 40 uL | align="center" align="center" valign="bottom" | 4.9 | align="center" align="center" valign="bottom" | 4.4 | align="center" align="center" valign="bottom" | 5.5 | align="center" align="center" valign="bottom" | 5.3 |- style="font-size:12pt" |style="font-weight:bold" height="15" valign="bottom" | ug total | align="center" align="center" valign="bottom" | |style="font-weight:bold" align="center" align="center" valign="bottom" | 9.3 | align="center" align="center" valign="bottom" | |style="font-weight:bold" align="center" align="center" valign="bottom" | 10.8 |- style="font-size:12pt" |style="font-weight:bold" height="30" valign="bottom" | Yield (Max ~80%) | align="center" valign="bottom" | |style="font-weight:bold" align="center" align="center" valign="bottom" | 71.18 | align="center" align="center" valign="bottom" | |style="font-weight:bold" align="center" align="center" valign="bottom" | 71.18 |} Scary that both of the columns yielded the same percent (I double checked the math). As a note, taking into account that the theoretical maximum yield is 80% (since we remove 20bp out of 100 on each strand), this is approximately a '''90% yield''' from the column. ===TBE Gel=== '''Gel Image''' [[Image:2014_07_09_DpnII.jpg|500px]] ==Lambda Exonuclease Digestion== #Reaction Table {| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext> |- style="background-color:#C5D9F1;font-size:12pt;font-weight:bold" | width="119" height="15" valign="bottom" | Sample | width="65" align="center" valign="bottom" | V6S2AB | width="65" align="center" valign="bottom" | V6S2CD | width="65" align="center" valign="bottom" | V6S3AB | width="65" align="center" valign="bottom" | V6S3CD |- style="font-size:12pt" |style="font-weight:bold" height="15" valign="bottom" | ng/uL | align="center" align="center" valign="bottom" | 122.4 | align="center" align="center" valign="bottom" | 110.7 | align="center" align="center" valign="bottom" | 137 | align="center" align="center" valign="bottom" | 133.5 |- style="background-color:#D9D9D9;font-size:12pt" |style="font-weight:bold" height="15" valign="bottom" | ug in 35 uL | align="center" align="center" valign="bottom" | 4.3 | align="center" align="center" valign="bottom" | 3.9 | align="center" align="center" valign="bottom" | 4.8 | align="center" align="center" valign="bottom" | 4.7 |- style="font-size:12pt" |style="font-weight:bold" height="15" valign="bottom" | uL sample | align="center" align="center" valign="bottom" | 35 | align="center" align="center" valign="bottom" | 35 | align="center" align="center" valign="bottom" | 35 | align="center" align="center" valign="bottom" | 35 |- style="background-color:#D9D9D9;font-size:12pt" |style="font-weight:bold" height="15" valign="bottom" | uL 10X buffer | align="center" align="center" valign="bottom" | 5 | align="center" align="center" valign="bottom" | 5 | align="center" align="center" valign="bottom" | 5 | align="center" align="center" valign="bottom" | 5 |- style="font-size:12pt" |style="font-weight:bold" height="30" valign="bottom" | uL Lambda Exonuclease (5 U/uL) | align="center" align="center" valign="bottom" | 3 | align="center" align="center" valign="bottom" | 3 | align="center" align="center" valign="bottom" | 3 | align="center" align="center" valign="bottom" | 3 |- style="background-color:#D9D9D9;font-size:12pt" |style="font-weight:bold" height="15" valign="bottom" | uL nfH2O | align="center" align="center" valign="bottom" | 7 | align="center" align="center" valign="bottom" | 7 | align="center" align="center" valign="bottom" | 7 | align="center" align="center" valign="bottom" | 7 |- style="font-size:12pt" |style="font-weight:bold" height="15" valign="bottom" | Total | align="center" align="center" valign="bottom" | 50 | align="center" align="center" valign="bottom" | 50 | align="center" align="center" valign="bottom" | 50 | align="center" align="center" valign="bottom" | 50 |} #Incubate for 1 hr at 37C #Heat inactivate for 10 minutes at 75C #Purify with [[Daniel:Protocols/ssDNA Column|Zymo column]], elute with 20 uL nfH2O ===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" | width="82" height="15" valign="bottom" | Sample | width="65" align="center" valign="bottom" | V6S2 | width="65" align="center" valign="bottom" | V6S3 |- style="font-size:12pt" |style="font-weight:bold" height="15" valign="bottom" | ng/uL ssDNA | align="center" align="center" valign="bottom" | 187.6 | align="center" align="center" valign="bottom" | 228.1 |- style="font-size:12pt" |style="font-weight:bold" height="15" valign="bottom" | ug ssDNA | align="center" align="center" valign="bottom" | 3.8 | align="center" align="center" valign="bottom" | 4.6 |- style="font-size:12pt" |style="font-weight:bold" height="30" valign="bottom" | Yield (Max ~50%) | align="center" align="center" valign="bottom" | 42.16 | align="center" align="center" valign="bottom" | 48.02 |} Again, since the theoretical maximum of this reaction is 50% (we eliminate 1 strand), this yields an effective 85% and 96% yield from the reaction/column, which is pretty good. [[Category:HRCP]] [[Category:20140708]] [[Category:Lambda Exo]]
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