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=Version 3 Oligos (Started [[Daniel:Notebook/ComboLock/2017-1-13|Thursday, Jan 13]])= [[Daniel:Notebook/ComboLock|Back to Calendar]] ==RCA Test-Adpaters== From [[Daniel:Notebook/ComboLock/2017-1-19|yesterday]]'s results I'm going to try the short RCA again, this time using the adapter regions as primers. I'll test using Lock0301 and Latch0301, since those should only amplify if circular product exists. <ol> <li>Rolling Circle Amplification</li> <ol type="A"> <li>Prepare 8.5X Master mix according to table below</li> <li>Add 12.5 uL master mix to each tube</li> <li>Add sample to new reaction tubes according to following 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="200" height="30" | Reagent | width="65" | uL Added | width="90" | Master Mix (8.5X) |- style="font-size:12pt" | height="15" valign="bottom" | Template | align="center" align="center" valign="bottom" | 5 | align="center" valign="bottom" | NA |- style="background-color:#BFBFBF;font-size:12pt" | height="15" valign="bottom" | RCA Primer (Latch0301 or Lock0301); 10 uM) | align="center" align="center" valign="bottom" | 2.5 | align="center" align="center" valign="bottom" | 21.25 |- style="font-size:12pt" | height="15" valign="bottom" | dNTP (1 mM) | align="center" align="center" valign="bottom" | 0.8 | align="center" align="center" valign="bottom" | 6.8 |- style="background-color:#BFBFBF;font-size:12pt" | height="15" valign="bottom" | 10X Phi29 Buffer | align="center" align="center" valign="bottom" | 2 | align="center" align="center" valign="bottom" | 17 |- style="font-size:12pt" | height="15" valign="bottom" | Phi29 | align="center" align="center" valign="bottom" | 1 | align="center" align="center" valign="bottom" | 8.5 |- style="background-color:#BFBFBF;font-size:12pt" | height="15" valign="bottom" | nfH2O | align="center" align="center" valign="bottom" | 8.7 | align="center" align="center" valign="bottom" | 73.95 |- style="font-size:12pt" | height="15" valign="bottom" | Total |style="font-weight:bold" align="center" align="center" valign="bottom" | 20 |style="font-weight:bold" align="center" align="center" valign="bottom" | 127.5 |} <li>Incubate at 37C for 30 minutes</li> <li>Incubate at 65C for 10 minutes;Hold at 10C until next step</li></ol> <li>Qubit Measure</li> <ol type="A"> <li>Mix dye to Qubit buffer; 10 uL dye in 1990 uL buffer (1:200 ratio per sample)</li> <li>Add 190 uL dye/buffer and 10 uL standard to standard tubes</li> <li>Add 199 uL dye/buffer and 1 uL sample to sample tubes</li> <li>Measure in Qubit</li> </ol></ol> ===Results=== {| 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="98" height="36" | Sample Name | width="65" | 1A | width="65" | 1B | width="65" | 1AL | width="65" | 1BL | width="65" | 4AL | width="65" | 4BL | width="65" | 1AK | width="65" | 1BK | width="65" | 4AK | width="60" | 4BK |- style="font-size:12pt" | height="30" | Sample Condition | align="center" | Sample-PreRCA | align="center" | Sample-PreRCA | align="center" | Sample | align="center" | Sample | align="center" | No PO4 (PCCB) | align="center" | No PO4 (PCCB) | align="center" | Sample | align="center" | Sample | align="center" | No PO4 (PCCB) | align="center" | No PO4 (PCCB) |- style="background-color:#BFBFBF;font-size:12pt" | height="15" | RCA Primer | align="center" | None | align="center" | None | align="center" | Latch0301 | align="center" | Latch0301 | align="center" | Latch0301 | align="center" | Latch0301 | align="center" | Primer6 | align="center" | Primer6 | align="center" | Primer6 | align="center" | Primer6 |- style="font-size:12pt" | height="15" | Conc (ng/uL) | align="center" align="center" | 0.81 | align="center" align="center" | 1.27 | align="center" align="center" | 1.76 | align="center" align="center" | 2.52 | align="center" align="center" | 2.22 | align="center" align="center" | 2.35 | align="center" align="center" | 0.25 | align="center" align="center" | 0.27 | align="center" align="center" | 0.37 | align="center" align="center" | 0.29 |- style="font-size:12pt" | height="30" valign="bottom" | Fold Change from Pre-RCA | align="center" | NA | align="center" | NA | align="center" align="center" | 2.2 | align="center" align="center" | 2.0 | align="center" align="center" | 2.7 | align="center" align="center" | 1.9 | align="center" align="center" | 0.3 | align="center" align="center" | 0.2 | align="center" align="center" | 0.5 | align="center" align="center" | 0.2 |} So this doesn't show anything definitive. It seems that the RCA didn't work at all with either of these. I'm going to try hexamers, which ought to amplify anything and everything (exponentially, actually) ==Random Hexamer RCA== RCA Test (again) using random hexamers. <ol> <li>Rolling Circle Amplification</li> <ol type="A"> <li>Prepare 4.5X Master mix according to table below</li> <li>Add 15 uL master mix to each tube</li> <li>Add sample to new reaction tubes according to following 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="200" height="30" | Reagent | width="65" | uL Added | width="90" | Master Mix (4.5X) |- style="font-size:12pt" | height="15" valign="bottom" | Template | align="center" align="center" valign="bottom" | 5 | align="center" valign="bottom" | NA |- style="background-color:#BFBFBF;font-size:12pt" | height="15" valign="bottom" | Random Hexamer (100 uM) | align="center" align="center" valign="bottom" | 2.5 | align="center" align="center" valign="bottom" | 11.25 |- style="font-size:12pt" | height="15" valign="bottom" | dNTP (1 mM) | align="center" align="center" valign="bottom" | 0.8 | align="center" align="center" valign="bottom" | 3.6 |- style="background-color:#BFBFBF;font-size:12pt" | height="15" valign="bottom" | 10X Phi29 Buffer | align="center" align="center" valign="bottom" | 2 | align="center" align="center" valign="bottom" | 9 |- style="font-size:12pt" | height="15" valign="bottom" | Phi29 | align="center" align="center" valign="bottom" | 1 | align="center" align="center" valign="bottom" | 4.5 |- style="background-color:#BFBFBF;font-size:12pt" | height="15" valign="bottom" | nfH2O | align="center" align="center" valign="bottom" | 8.7 | align="center" align="center" valign="bottom" | 39.15 |- style="font-size:12pt" | height="15" valign="bottom" | Total |style="font-weight:bold" align="center" align="center" valign="bottom" | 20 |style="font-weight:bold" align="center" align="center" valign="bottom" | 67.5 |} <li>Incubate at 37C for 30 minutes</li> <li>Incubate at 65C for 10 minutes;Hold at 10C until next step</li></ol> <li>Qubit Measure</li> <ol type="A"> <li>Mix dye to Qubit buffer; 10 uL dye in 1990 uL buffer (1:200 ratio per sample)</li> <li>Add 190 uL dye/buffer and 10 uL standard to standard tubes</li> <li>Add 199 uL dye/buffer and 1 uL sample to sample tubes</li> <li>Wait 5 minutes to allow dye saturation</li> <li>Measure in Qubit</li> </ol></ol> ===Results=== {| 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="130" height="36" | Sample Name | width="65" | 1A | width="65" | 1B | width="85" | 1A6 | width="85" | 1B6 | width="85" | 4A6 | width="85" | 4B6 |- style="font-size:12pt" | height="30" | Sample Condition | align="center" | Sample-PreRCA | align="center" | Sample-PreRCA | align="center" | Sample | align="center" | Sample | align="center" | No PO4 (PCCB) | align="center" | No PO4 (PCCB) |- style="background-color:#BFBFBF;font-size:12pt" | height="30" | RCA Primer | align="center" | None | align="center" | None | align="center" | Random Hexamer | align="center" | Random Hexamer | align="center" | Random Hexamer | align="center" | Random Hexamer |- style="font-size:12pt" | height="15" | Conc (ng/uL) | align="center" align="center" | 0.81 | align="center" align="center" | 1.27 | align="center" align="center" | 16.6 | align="center" align="center" | 16.5 | align="center" align="center" | 15.4 | align="center" align="center" | 20.5 |- style="font-size:12pt" | height="30" valign="bottom" | Fold Change from Pre-RCA | align="center" | NA | align="center" | NA | align="center" align="center" | 20.5 | align="center" align="center" | 13.0 | align="center" align="center" | 19.0 | align="center" align="center" | 16.1 |} So either everything amplified (weakly) or nothing amplified. I think it's everything weakly. ==qPCR-RCA Results== Cause, why not? This will test the (hopefully) fold changes as well as confirm the product result. To that end, I'll use the primer12RC/AmpR.Ind1 pair, since that gives a product of 150 (and should only occur on ligated product). <ol> <li>qPCR</li> <ol type="A"> <li>Make 15.2X qPCR master mix according to following recipe</li> <ol type="a"> <li>319.2 uL nfH2O</li> <li>380 uL SYBR Fast</li> <li>15.2 uL 10 uM primer6RC</li> <li>15.2 uL 10 uM AmpR.Ind21</li> </ol> <li>Add 48 uL master mix to each well</li> <li>Add 2 uL sample 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="130" height="45" | Sample | width="120" | Condition | width="120" | RCA Primer | width="65" | Lane | width="65" | Sample Vol (uL) | width="85" | 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-PreRCA | align="center" | None | 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 4A | align="center" | No P04 (PCCB)-PreRCA | align="center" | None | 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 1AL | align="center" | Sample | align="center" | Latch0301 | 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:#DA9694;font-size:12pt" |style="font-weight:bold" height="15" valign="bottom" | Sample 1BL | align="center" valign="bottom" | Sample | align="center" valign="bottom" | Latch0301 | 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 4AL | align="center" | No PCCB PO4 | align="center" | Latch0301 | 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:#DA9694;font-size:12pt" |style="font-weight:bold" height="15" | Sample 4BL | align="center" | No PCCB PO4 | align="center" valign="bottom" | Latch0301 | 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 1AK | align="center" | Sample | align="center" | Primer6 | 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:#C4D79B;font-size:12pt" |style="font-weight:bold" height="15" | Sample 1BK | align="center" valign="bottom" | Sample | align="center" | Primer6 | 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 4AK | align="center" | No PCCB PO4 | align="center" | Primer6 | 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" | 21 | align="center" align="center" | 50 |- style="background-color:#C4D79B;font-size:12pt" |style="font-weight:bold" height="15" | Sample 4BK | align="center" | No PCCB PO4 | align="center" | Primer6 | align="center" | H2 | 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" | Sample 1A6 | align="center" | Sample | align="center" | Random Hexamer | align="center" | H3 | 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:#B1A0C7;font-size:12pt" |style="font-weight:bold" height="30" | Sample 1B6 | align="center" | Sample | align="center" | Random Hexamer | align="center" | H4 | 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" | Sample 4A6 | align="center" | No PCCB PO4 | align="center" | Random Hexamer | align="center" | H5 | 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:#B1A0C7;font-size:12pt" |style="font-weight:bold" height="30" | Sample 4B6 | align="center" | No PCCB PO4 | align="center" | Random Hexamer | align="center" | H6 | 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" | height="30" valign="bottom" | NTC | align="center" valign="bottom" | qPCR Primers Only | align="center" valign="bottom" | None | align="center" | H7 | align="center" align="center" | 0 | 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> <li>Held at 4C overnight after PCR finished;Gel run 1.21.17</li> </ol></ol> <li>TBE Gel</li> <ol type="A"> <li>Mix 160 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 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:20170120-qPCR-v3Oligos-RCA-QC.png|qPCR curve File:2017-01-23-v3Oligos-RCA-QC1.png|Gel image 1 File:2017-01-23-v3Oligos-RCA-QC2.png|Gel image 2 </gallery> [[Category:ComboLock]] [[Category:20170113]]
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