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=Alu Control ([[Daniel:Notebook/HiResChrPaint/2014-1-8|Started 01/08/2014]])= [[Daniel:Notebook/HiResChrPaint|Back to Calendar]] ==Dye Coupling== 1. Add 100 uL DMSO to vial containing ULS dye (stable for 6 months at 4C) *For AlexaFluor 488 label using 5 uL DMSO (stable for 1 month at 4C) ***VORTEX THESE SOLUTIONS '''VIGOROUSLY''' BEFORE USE '''EVERY TIME'''! 2. Resuspend DNA in 20 uL labeling buffer (Component C) 3. Denature DNA at 95C for 5 minutes and snap cool on ice; centrifuge briefly 4. Add appropriate volume of dye to sample (see table 2); if necessary add labeling buffer to bring volume to 25 uL 5. Incubate at 80C for 15 minutes; stop the reaction by plunging the tube into an ice bath 5-1 Add 5 uL nfH2O, mix thoroughly, then extract 5 uL and put into 45 uL nfH2O (pre-column sample) 6. Purify using a [[Daniel:Protocols/CentriSep|Centri-Sep column]] ===Labeling Results=== {| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext> |- style="font-size:12pt" | align="center" width="65" height="15" valign="bottom" | |style="background-color:#8DB4E2;font-weight:bold" width="65" colspan="4" align="center" valign="bottom" | Pre Column |style="background-color:#E6B8B7;font-weight:bold" width="65" colspan="4" align="center" valign="bottom" | Post Column | align="center" width="65" align="center" valign="bottom" | | align="center" width="65" align="center" valign="bottom" | |- style="font-size:12pt;font-weight:bold" align="center" |style="background-color:#D9D9D9" height="45" | Sample |style="background-color:#8DB4E2" | ng/uL ssDNA |style="background-color:#8DB4E2" | pmol/uL dye |style="background-color:#8DB4E2" | pmol/uL ss probe |style="background-color:#8DB4E2" | base:dye |style="background-color:#E6B8B7" | ng/uL ssDNA |style="background-color:#E6B8B7" | pmol/uL dye |style="background-color:#E6B8B7" | pmol/uL ss probe |style="background-color:#E6B8B7" | base:dye |style="background-color:#D8E4BC" | DNA retention (%) |style="background-color:#D8E4BC" | Dye retention (%) |- style="font-size:12pt" align="center" |style="background-color:#D9D9D9;font-weight:bold" height="15" | Cot1-A |style="background-color:#8DB4E2" align="center" | 0 |style="background-color:#8DB4E2" align="center" | 0.9 |style="background-color:#8DB4E2" align="center" | 0.0 |style="background-color:#8DB4E2" align="center" | 0.0 |style="background-color:#E6B8B7" align="center" | 3.3 |style="background-color:#E6B8B7" align="center" | 0.3 |style="background-color:#E6B8B7" align="center" | 0.1 |style="background-color:#E6B8B7" align="center" | 33 |style="background-color:#D8E4BC" | NA |style="background-color:#D8E4BC" align="center" | 2.22 |- style="font-size:12pt" align="center" |style="background-color:#D9D9D9;font-weight:bold" height="15" | Cot1-B |style="background-color:#8DB4E2" align="center" | 1.4 |style="background-color:#8DB4E2" align="center" | 0.8 |style="background-color:#8DB4E2" align="center" | 0.0 |style="background-color:#8DB4E2" align="center" | 5 |style="background-color:#E6B8B7" align="center" | 13.1 |style="background-color:#E6B8B7" align="center" | 0.3 |style="background-color:#E6B8B7" align="center" | 0.2 |style="background-color:#E6B8B7" align="center" | 132 |style="background-color:#D8E4BC" align="center" | 62.38 |style="background-color:#D8E4BC" align="center" | 2.50 |- style="font-size:12pt" align="center" |style="background-color:#D9D9D9;font-weight:bold" height="15" | Blank |style="background-color:#8DB4E2" align="center" | 0 |style="background-color:#8DB4E2" align="center" | 0.8 |style="background-color:#8DB4E2" align="center" | 0.0 |style="background-color:#8DB4E2" align="center" | 0.0 |style="background-color:#E6B8B7" align="center" | 0 |style="background-color:#E6B8B7" align="center" | 0 |style="background-color:#E6B8B7" align="center" | 0.0 |style="background-color:#E6B8B7" | NA |style="background-color:#D8E4BC" | NA |style="background-color:#D8E4BC" align="center" | 0.00 |- style="font-size:12pt" align="center" |style="background-color:#D9D9D9;font-weight:bold" height="15" | DNA Only |style="background-color:#8DB4E2" align="center" | 2.1 |style="background-color:#8DB4E2" align="center" | 0 |style="background-color:#8DB4E2" align="center" | 0.0 |style="background-color:#8DB4E2" | NA |style="background-color:#E6B8B7" align="center" | 19.4 |style="background-color:#E6B8B7" align="center" | 0 |style="background-color:#E6B8B7" align="center" | 0.3 |style="background-color:#E6B8B7" | NA |style="background-color:#D8E4BC" align="center" | 61.59 |style="background-color:#D8E4BC" | NA |} '''S'''o these are pretty expected results, at least from the controls. The DNA only sample showed similar DNA retention to the mixed sample (but with no dye retention of course). The blank sample, which had dye but no DNA, had no DNA retention and no dye retention, indicating that the ULS dye did not move quickly through the column. The samples themselves, however, are harder to piece out. '''O'''ne problem is that the pre-column samples were diluted 1:10 before being measured in the nanodrop. So I think that the Cot1-A sample (precipitated using EtOH precipitation) had too little starter material to show up on the pre-column sample. Hence, why there was magically DNA after the column. '''H'''owever, this now brings up an interesting point. Since there was 30 uL in the sample this was drawn from, and it was diluted 1:10, we can multiply back the 1.4 ng/uL in the Cot1-B sample to get that ~420 ng of starting material were recovered. We can also, assuming a ~60% DNA recovery on the column (since both other DNA samples retained ~60% on the column), back calculate that the starting material was 3.3*20 uL /0.6 = 110 ng starting material. It follows then that the EtOH precipitated sample had on the order of 1/4 as much DNA as the vacuum dried sample.
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