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=Extended Latch Test (Started [[Daniel:Notebook/ComboLock/2016-11-29|Nov 29]])= [[Daniel:Notebook/ComboLock|Back to Calendar]] ===Sample Matrix=== {| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext> |- style="background-color:#CCC0DA;font-size:12pt;font-weight:bold" align="center" | width="150" height="34" | Sample | width="120" | Condition |- style="font-size:12pt" | height="15" valign="bottom" | Sample 1 (AB) | align="center" valign="bottom" | Normal |- style="background-color:#BFBFBF;font-size:12pt" | height="15" valign="bottom" | Sample 2 (AB) | align="center" valign="bottom" | No Template |- style="font-size:12pt" | height="15" valign="bottom" | Sample 3 (AB) | align="center" valign="bottom" | No phosphate activation |- style="background-color:#BFBFBF;font-size:12pt" | height="15" valign="bottom" | Sample 4 (AB) | align="center" valign="bottom" | No C Probe |} ==Beads smear test== One thing I have been doing in the last few weeks is being careful to agitate the beads and get a few into the RCA reaction. I've been doing this in case the circular product is in some way still attached to the beads. However, it could explain why there are smears in both products. I'm going to test a few scenarios to examine this hypothesis. *Gel run of unagitated (not containing beads) and agitated (containing beads) media *Repeat RCA using unagitated supernatant from the exonuclease reaction ===Samples List=== {| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext> |- style="background-color:#92CDDC;font-size:12pt;font-weight:bold" align="center" | width="50" height="50" | Lane | width="150" | Sample | width="120" | Condition |- style="font-size:12pt" | align="center" height="15" valign="bottom" | 2 | align="center" valign="bottom" | 25bp Ladder | align="center" valign="bottom" | NA |- style="background-color:#BFBFBF;font-size:12pt" | align="center" height="15" valign="bottom" | 3 | align="center" valign="bottom" | Sample A | align="center" valign="bottom" | Supernatant |- style="font-size:12pt" | align="center" height="15" valign="bottom" | 4 | align="center" valign="bottom" | Sample B | align="center" valign="bottom" | Supernatant |- style="background-color:#BFBFBF;font-size:12pt" | align="center" height="15" valign="bottom" | 5 | align="center" valign="bottom" | Sample A | align="center" valign="bottom" | Beads+ |- style="font-size:12pt" | align="center" height="15" valign="bottom" | 6 | align="center" valign="bottom" | Sample B | align="center" valign="bottom" | Beads+ |- style="background-color:#BFBFBF;font-size:12pt" | align="center" height="15" valign="bottom" | 7 | align="center" valign="bottom" | No Template A | align="center" valign="bottom" | Supernatant |- style="font-size:12pt" | align="center" height="15" valign="bottom" | 8 | align="center" valign="bottom" | No Template B | align="center" valign="bottom" | Supernatant |- style="background-color:#BFBFBF;font-size:12pt" | align="center" height="15" valign="bottom" | 9 | align="center" valign="bottom" | No Template A | align="center" valign="bottom" | Beads+ |- style="font-size:12pt" | align="center" height="15" valign="bottom" | 10 | align="center" valign="bottom" | No Template B | align="center" valign="bottom" | Beads+ |} <ol> <li>TBE Gel</li> <ol type="A"> <li>Mix 80 uL TBE, 20 uL 6x loading dye</li> <li>Aliquot 10 uL per sample/ladder lane onto parafilm</li> <li>Add 2 uL of sample or ladder to correct drop</li> <li>Load 10 uL in to well</li> <li>Run gel for 25 minutes at 225V</li> <li>Open gel and stain with 3 uL SYBR Gold for 3 minutes</li> <li>Rinse gel and image in gel doc</li> </ol></ol> [[Image:2016-12-01-ExtLatch-QC1.png|500px]] Doesn't really show anything, the concentration of both is too low. ==No Beads RCA== <ol start="7"> <li>Rolling Circle Amplification</li> <ol type="A"> <li>Prepare 9X master mix according to table below</li> {| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext> |- style="background-color:#C4D79B;font-size:12pt;font-weight:bold" align="center" | width="160" height="30" | Reagent | width="94" | Stock Concentration | width="77" | Final Conc/Amt | width="74" | uL Added in 20 uL Rxn | width="65" | Master Mix (9X) |- style="font-size:12pt" | height="15" valign="bottom" | Template | align="center" valign="bottom" | NA | align="center" valign="bottom" | NA | align="center" align="center" valign="bottom" | 5 | align="center" align="center" valign="bottom" | NA |- style="background-color:#BFBFBF;font-size:12pt" | height="15" valign="bottom" | RCA Primer (RCA-CL-LinkerLRC-v01) | align="center" valign="bottom" | 10 uM | align="center" valign="bottom" | 2 uM | align="center" align="center" valign="bottom" | 4 | align="center" align="center" valign="bottom" | 36 |- style="font-size:12pt" | height="15" valign="bottom" | dNTP | align="center" valign="bottom" | 1 mM | align="center" valign="bottom" | 50 uM | align="center" align="center" valign="bottom" | 1 | align="center" align="center" valign="bottom" | 9 |- style="background-color:#BFBFBF;font-size:12pt" | height="15" valign="bottom" | 10X Buffer | align="center" valign="bottom" | 10X | align="center" valign="bottom" | 1X | align="center" align="center" valign="bottom" | 2 | align="center" align="center" valign="bottom" | 18 |- style="font-size:12pt" | height="15" valign="bottom" | Phi29 (NEB) | align="center" valign="bottom" | 10 U/uL | align="center" valign="bottom" | 10 U | align="center" align="center" valign="bottom" | 1 | align="center" align="center" valign="bottom" | 9 |- style="background-color:#BFBFBF;font-size:12pt" | height="15" valign="bottom" | nfH2O | align="center" valign="bottom" | NA | align="center" valign="bottom" | NA | align="center" align="center" valign="bottom" | 7 | align="center" align="center" valign="bottom" | 63 |- style="font-size:12pt" | height="15" valign="bottom" | Total | align="center" align="center" valign="bottom" | | align="center" align="center" valign="bottom" | |style="font-weight:bold" align="center" align="center" valign="bottom" | 20 |style="font-weight:bold" align="center" align="center" valign="bottom" | 135 |} <li>Add 15 uL master mix to each tube</li> <li>Add 5 uL sample to correct reaction tubes</li> <li>Incubate at 37C for 3 hours</li> <li>Heat kill by incubating at 65C for 10 minutes</li></ol> <li>qPCR</li> <ol type="A"> <li>Make master mix according to following recipe</li> <ol type="a"> <li>359.1 uL nfH2O</li> <li>427.5 uL SYBR Fast</li> <li>17.1 uL 10 uM AmpF</li> </ol> <li>Add 47 uL master mix to each well</li> <li>Add 2 uL sample and 1 uL appropriate Reverse Primer (1 uM) according to table</li> {| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext> |- style="background-color:#92CDDC;font-size:12pt;font-weight:bold" align="center" | width="85" height="30" | Sample | width="125" | Condition | width="80" | RCA | width="85" | Lane | width="85" | AmpR Index | width="85" | Sample Vol (uL) | width="86" | 2X Kapa SYBR qPCR MM | width="84" | 10 uM Forward Primer | width="83" | 10 uM Reverse Primer | width="85" | H2O | width="86" | Total Volume (uL) |- style="font-size:12pt" |style="font-weight:bold" height="15" | Sample 1A | align="center" | Sample | align="center" | Y | align="center" | A1 | align="center" align="center" | 20 | align="center" align="center" | 2 | align="center" align="center" | 25 | align="center" align="center" | 1 | align="center" align="center" | 1 | align="center" align="center" | 21 | align="center" align="center" | 50 |- style="background-color:#BFBFBF;font-size:12pt" |style="font-weight:bold" height="15" | Sample 1B | align="center" | Sample | align="center" | Y | align="center" | A2 | align="center" align="center" | 20 | align="center" align="center" | 2 | align="center" align="center" | 25 | align="center" align="center" | 1 | align="center" align="center" | 1 | align="center" align="center" | 21 | align="center" align="center" | 50 |- style="font-size:12pt" |style="font-weight:bold" height="15" | Sample 2A | align="center" | No Template | align="center" | Y | align="center" | A3 | align="center" align="center" | 21 | align="center" align="center" | 2 | align="center" align="center" | 25 | align="center" align="center" | 1 | align="center" align="center" | 1 | align="center" align="center" | 21 | align="center" align="center" | 50 |- style="background-color:#BFBFBF;font-size:12pt" |style="font-weight:bold" height="15" valign="bottom" | Sample 2B | align="center" valign="bottom" | No Template | align="center" valign="bottom" | Y | align="center" | A4 | align="center" align="center" | 21 | align="center" align="center" | 2 | align="center" align="center" | 25 | align="center" align="center" | 1 | align="center" align="center" | 1 | align="center" align="center" | 21 | align="center" align="center" | 50 |- style="font-size:12pt" |style="font-weight:bold" height="15" | Sample 3A | align="center" | No Phosphate | align="center" | Y | align="center" | A5 | align="center" align="center" | 22 | align="center" align="center" | 2 | align="center" align="center" | 25 | align="center" align="center" | 1 | align="center" align="center" | 1 | align="center" align="center" | 21 | align="center" align="center" | 50 |- style="background-color:#BFBFBF;font-size:12pt" |style="font-weight:bold" height="15" | Sample 3B | align="center" | No Phosphate | align="center" | Y | align="center" | A6 | align="center" align="center" | 22 | align="center" align="center" | 2 | align="center" align="center" | 25 | align="center" align="center" | 1 | align="center" align="center" | 1 | align="center" align="center" | 21 | align="center" align="center" | 50 |- style="font-size:12pt" |style="font-weight:bold" height="15" | Sample 4A | align="center" | No C Probe | align="center" | Y | align="center" | A7 | align="center" align="center" | 23 | align="center" align="center" | 2 | align="center" align="center" | 25 | align="center" align="center" | 1 | align="center" align="center" | 1 | align="center" align="center" | 21 | align="center" align="center" | 50 |- style="background-color:#BFBFBF;font-size:12pt" |style="font-weight:bold" height="15" | Sample 4B | align="center" | No C Probe | align="center" | Y | align="center" | A8 | align="center" align="center" | 23 | align="center" align="center" | 2 | align="center" align="center" | 25 | align="center" align="center" | 1 | align="center" align="center" | 1 | align="center" align="center" | 21 | align="center" align="center" | 50 |- style="font-size:12pt" |style="font-weight:bold" height="15" | Sample 1A | align="center" | Sample | align="center" | N | align="center" | A1 | align="center" align="center" | 24 | align="center" align="center" | 2 | align="center" align="center" | 25 | align="center" align="center" | 1 | align="center" align="center" | 1 | align="center" align="center" | 21 | align="center" align="center" | 50 |- style="background-color:#BFBFBF;font-size:12pt" |style="font-weight:bold" height="15" | Sample 1B | align="center" | Sample | align="center" | N | align="center" | A2 | align="center" align="center" | 24 | align="center" align="center" | 2 | align="center" align="center" | 25 | align="center" align="center" | 1 | align="center" align="center" | 1 | align="center" align="center" | 21 | align="center" align="center" | 50 |- style="font-size:12pt" |style="font-weight:bold" height="15" | Sample 2A | align="center" | No Template | align="center" | N | align="center" | A3 | align="center" align="center" | 25 | align="center" align="center" | 2 | align="center" align="center" | 25 | align="center" align="center" | 1 | align="center" align="center" | 1 | align="center" align="center" | 21 | align="center" align="center" | 50 |- style="background-color:#BFBFBF;font-size:12pt" |style="font-weight:bold" height="15" valign="bottom" | Sample 2B | align="center" valign="bottom" | No Template | align="center" valign="bottom" | N | align="center" | A4 | align="center" align="center" | 25 | align="center" align="center" | 2 | align="center" align="center" | 25 | align="center" align="center" | 1 | align="center" align="center" | 1 | align="center" align="center" | 21 | align="center" align="center" | 50 |- style="font-size:12pt" |style="font-weight:bold" height="15" | Sample 3A | align="center" | No Phosphate | align="center" | N | align="center" | A5 | align="center" align="center" | 26 | align="center" align="center" | 2 | align="center" align="center" | 25 | align="center" align="center" | 1 | align="center" align="center" | 1 | align="center" align="center" | 21 | align="center" align="center" | 50 |- style="background-color:#BFBFBF;font-size:12pt" |style="font-weight:bold" height="15" | Sample 3B | align="center" | No Phosphate | align="center" | N | align="center" | A6 | align="center" align="center" | 26 | align="center" align="center" | 2 | align="center" align="center" | 25 | align="center" align="center" | 1 | align="center" align="center" | 1 | align="center" align="center" | 21 | align="center" align="center" | 50 |- style="font-size:12pt" |style="font-weight:bold" height="15" | Sample 4A | align="center" | No C Probe | align="center" | N | align="center" | A7 | align="center" align="center" | 27 | align="center" align="center" | 2 | align="center" align="center" | 25 | align="center" align="center" | 1 | align="center" align="center" | 1 | align="center" align="center" | 21 | align="center" align="center" | 50 |- style="background-color:#BFBFBF;font-size:12pt" |style="font-weight:bold" height="15" | Sample 4B | align="center" | No C Probe | align="center" | N | align="center" | A8 | align="center" align="center" | 27 | align="center" align="center" | 2 | align="center" align="center" | 25 | align="center" align="center" | 1 | align="center" align="center" | 1 | align="center" align="center" | 21 | align="center" align="center" | 50 |- style="font-size:12pt" |style="font-weight:bold" height="15" valign="bottom" | NTC | align="center" valign="bottom" | qPCR Primers Only | align="center" valign="bottom" | NA | align="center" | H1 | align="center" align="center" | 28 | align="center" align="center" | 2 | align="center" align="center" | 25 | align="center" align="center" | 1 | align="center" align="center" | 1 | align="center" align="center" | 23 | align="center" align="center" | 50 |} <li>qPCR Cycles</li> <ol type="a"> <li>95C 3 min</li> <li>95C 3 sec</li> <li>55C 30 sec</li> <li>72C 20 sec</li> <li>plate read</li> <li>goto b x24</li> <li>72C 2 min</li> <li>16C hold</li> </ol></ol> <li>TBE Gel</li> <ol type="A"> <li>Mix 200 uL TBE, 50 uL 6x loading dye</li> <li>Aliquot 10 uL per sample/ladder lane onto parafilm</li> <li>Add 2 uL of sample or ladder to correct drop</li> <li>Load 10 uL in to well</li> <li>Run gel for 22 minutes at 250V</li> <li>Open gel and stain with 3 uL SYBR Gold for 3 minutes</li> <li>Rinse gel and image in gel doc</li> </ol></ol> ===Results=== <gallery perrow=2 heights=300px widths=300px> File:20161201-qPCR-ExtLatch-QC2.png|qPCR image File:2016-12-01-ExtLatch-QC2-PostRCA.png|Gel image (RCA +) File:2016-12-01-ExtLatch-QC2-PreRCA.png|Gel image (Before RCA) </gallery> ==Adapter Amplification== <ol> <li>qPCR</li> <ol type="A"> <li>Make master mix according to following recipe</li> <ol type="a"> <li>359.1 uL nfH2O</li> <li>427.5 uL SYBR Fast</li> <li>17.1 uL 10 uM pimer24RC</li> <li>17.1 uL 10 uM pimer12</li> </ol> <li>Add 48 uL master mix to each well</li> <li>Add 2 uL sample and 1 uL appropriate Reverse Primer (1 uM) according to table</li> {| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext> |- style="background-color:#92CDDC;font-size:12pt;font-weight:bold" align="center" | width="100" height="45" | Sample | width="150" | Condition | width="50" | Lane | width="65" | Sample Vol (uL) | width="65" | 2X Kapa SYBR qPCR MM | width="65" | 10 uM Forward Primer | width="65" | 10 uM Reverse Primer | width="65" | H2O | width="65" | Total Volume (uL) |- style="font-size:12pt" |style="font-weight:bold" height="15" | Sample 1A | align="center" | Sample | align="center" | A1 | align="center" align="center" | 2 | align="center" align="center" | 25 | align="center" align="center" | 1 | align="center" align="center" | 1 | align="center" align="center" | 21 | align="center" align="center" | 50 |- style="background-color:#BFBFBF;font-size:12pt" |style="font-weight:bold" height="15" | Sample 1B | align="center" | Sample | align="center" | A2 | align="center" align="center" | 2 | align="center" align="center" | 25 | align="center" align="center" | 1 | align="center" align="center" | 1 | align="center" align="center" | 21 | align="center" align="center" | 50 |- style="font-size:12pt" |style="font-weight:bold" height="15" | Sample 2A | align="center" | No Template | align="center" | A3 | align="center" align="center" | 2 | align="center" align="center" | 25 | align="center" align="center" | 1 | align="center" align="center" | 1 | align="center" align="center" | 21 | align="center" align="center" | 50 |- style="background-color:#BFBFBF;font-size:12pt" |style="font-weight:bold" height="15" valign="bottom" | Sample 2B | align="center" valign="bottom" | No Template | align="center" | A4 | align="center" align="center" | 2 | align="center" align="center" | 25 | align="center" align="center" | 1 | align="center" align="center" | 1 | align="center" align="center" | 21 | align="center" align="center" | 50 |- style="font-size:12pt" |style="font-weight:bold" height="15" | Sample 3A | align="center" | No Phosphate | align="center" | A5 | align="center" align="center" | 2 | align="center" align="center" | 25 | align="center" align="center" | 1 | align="center" align="center" | 1 | align="center" align="center" | 21 | align="center" align="center" | 50 |- style="background-color:#BFBFBF;font-size:12pt" |style="font-weight:bold" height="15" | Sample 3B | align="center" | No Phosphate | align="center" | A6 | align="center" align="center" | 2 | align="center" align="center" | 25 | align="center" align="center" | 1 | align="center" align="center" | 1 | align="center" align="center" | 21 | align="center" align="center" | 50 |- style="font-size:12pt" |style="font-weight:bold" height="15" | Sample 4A | align="center" | No C Probe | align="center" | A7 | align="center" align="center" | 2 | align="center" align="center" | 25 | align="center" align="center" | 1 | align="center" align="center" | 1 | align="center" align="center" | 21 | align="center" align="center" | 50 |- style="background-color:#BFBFBF;font-size:12pt" |style="font-weight:bold" height="15" | Sample 4B | align="center" | No C Probe | align="center" | A8 | align="center" align="center" | 2 | align="center" align="center" | 25 | align="center" align="center" | 1 | align="center" align="center" | 1 | align="center" align="center" | 21 | align="center" align="center" | 50 |- style="font-size:12pt" |style="font-weight:bold" height="15" | Sample 1A | align="center" | Sample | align="center" | D1 | align="center" align="center" | 2 | align="center" align="center" | 25 | align="center" align="center" | 1 | align="center" align="center" | 1 | align="center" align="center" | 21 | align="center" align="center" | 50 |- style="background-color:#BFBFBF;font-size:12pt" |style="font-weight:bold" height="15" | Sample 1B | align="center" | Sample | align="center" | D2 | align="center" align="center" | 2 | align="center" align="center" | 25 | align="center" align="center" | 1 | align="center" align="center" | 1 | align="center" align="center" | 21 | align="center" align="center" | 50 |- style="font-size:12pt" |style="font-weight:bold" height="15" | Sample 2A | align="center" | No Template | align="center" | D3 | align="center" align="center" | 2 | align="center" align="center" | 25 | align="center" align="center" | 1 | align="center" align="center" | 1 | align="center" align="center" | 21 | align="center" align="center" | 50 |- style="background-color:#BFBFBF;font-size:12pt" |style="font-weight:bold" height="15" valign="bottom" | Sample 2B | align="center" valign="bottom" | No Template | align="center" | D4 | align="center" align="center" | 2 | align="center" align="center" | 25 | align="center" align="center" | 1 | align="center" align="center" | 1 | align="center" align="center" | 21 | align="center" align="center" | 50 |- style="font-size:12pt" |style="font-weight:bold" height="15" | Sample 3A | align="center" | No Phosphate | align="center" | D5 | align="center" align="center" | 2 | align="center" align="center" | 25 | align="center" align="center" | 1 | align="center" align="center" | 1 | align="center" align="center" | 21 | align="center" align="center" | 50 |- style="background-color:#BFBFBF;font-size:12pt" |style="font-weight:bold" height="15" | Sample 3B | align="center" | No Phosphate | align="center" | D6 | align="center" align="center" | 2 | align="center" align="center" | 25 | align="center" align="center" | 1 | align="center" align="center" | 1 | align="center" align="center" | 21 | align="center" align="center" | 50 |- style="font-size:12pt" |style="font-weight:bold" height="15" | Sample 4A | align="center" | No C Probe | align="center" | D7 | align="center" align="center" | 2 | align="center" align="center" | 25 | align="center" align="center" | 1 | align="center" align="center" | 1 | align="center" align="center" | 21 | align="center" align="center" | 50 |- style="background-color:#BFBFBF;font-size:12pt" |style="font-weight:bold" height="15" | Sample 4B | align="center" | No C Probe | align="center" | D8 | align="center" align="center" | 2 | align="center" align="center" | 25 | align="center" align="center" | 1 | align="center" align="center" | 1 | align="center" align="center" | 21 | align="center" align="center" | 50 |- style="font-size:12pt" |style="font-weight:bold" height="30" valign="bottom" | NTC | align="center" valign="bottom" | qPCR Primers Only | align="center" | H1 | align="center" align="center" | 2 | align="center" align="center" | 25 | align="center" align="center" | 1 | align="center" align="center" | 1 | align="center" align="center" | 23 | align="center" align="center" | 50 |} <li>qPCR Cycles</li> <ol type="a"> <li>95C 3 min</li> <li>95C 3 sec</li> <li>55C 30 sec</li> <li>72C 20 sec</li> <li>plate read</li> <li>goto b x24</li> <li>72C 2 min</li> <li>16C hold</li> </ol></ol> <li>TBE Gel</li> <ol type="A"> <li>Mix 176 uL TBE, 44 uL 6x loading dye</li> <li>Aliquot 10 uL per sample/ladder lane onto parafilm</li> <li>Add 2 uL of sample or ladder to correct drop</li> <li>Load 10 uL in to well</li> <li>Run gel for 22 minutes at 250V</li> <li>Open gel and stain with 3 uL SYBR Gold for 3 minutes</li> <li>Rinse gel and image in gel doc</li> </ol></ol> ===Results=== <gallery perrow=3 heights=300px widths=300px> File:20161201-qPCR-ExtLatch-AdpaterAmp.png|qPCR curves File:2016-12-02-ExtLatch-Adapter-PostRCA.png|Gel image post RCA File:2016-12-02-ExtLatch-Adapter-PreRCA.png|Gel image pre-RCA </gallery> [[Category:ComboLock]] [[Category:20161129]]
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