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AlanFung:LabNotes/Capturing/2014-1-23
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==Capturing ppMALAT1 template w ppMALT1 ordered form IDT== ==TBU GEL to verify probe template size== *Aim for 100ng/band *10bp Ladder *ppMALAT1 (92nt, 200uM=6072ng/ul) *MALAT1 Template (45nt, 100uM=1485ng/ul) *ppillumina_155 (99nt, 100uM=3267ng/ul) {| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8<\hiddentext> |- align="center" | width="274" height="13" | Sample | width="75" | ng/ul |style="background-color:#EBF1DE" width="75" | ul | width="273" | ng/ul | width="147" | ul |style="background-color:#EBF1DE" width="75" | H2O |- align="center" | height="13" | ppMALAT1 | align="center" | 6072 |style="background-color:#EBF1DE" align="center" | 0.40 | align="center" | 20 | align="center" | 120 |style="background-color:#EBF1DE" align="center" | 119.60 |- align="center" | height="13" | MALAT1 Template | align="center" | 1485 |style="background-color:#EBF1DE" align="center" | 0.40 | align="center" | 20 | align="center" | 30 |style="background-color:#EBF1DE" align="center" | 29.6 |- align="center" | height="13" | ppillumina_155 | align="center" | 3267 |style="background-color:#EBF1DE" align="center" | 0.40 | align="center" | 20 | align="center" | 65 |style="background-color:#EBF1DE" align="center" | 64.6 |- align="center" | height="13" | 10BP | align="center" | 1000 |style="background-color:#EBF1DE" align="center" | 0.40 | align="center" | 20 | align="center" | 20 |style="background-color:#EBF1DE" align="center" | 19.6 |} *Pre-run the gel for 30 min at 200V *Dilute sample to 20ng/uL *Denature samples and ladder at 75C for 7 min, then quickly transfer to cooling rack, wait for 5 mins *Flush the wells very well with P1000 & P200 pipette to remove all urea. *NOTE: DO NOT TOUCH THE TUBE WITH YOUR HANDS IN THE FOLLOWING STEP, GRAB THE CAP *Load the samples into the wells, run at 200V for 30 min. *Stain gel with 3uL SYBR Gold in ~100 mL of TBE in clean tray. *Place tray on an orbital shaker for ~5 min. [[File:ZhangLab_2 2014-01-23 13hr 49min.jpg|400px]] *1-ppMALAT1, 2-MALAT1_template, 3-illumina_155pp ==ssDNA Qubit to quantify probe and template== *ppMALAT1 (4.78ng/ul) /(0.4/120) = 1434ng/ul vs 6072ng/ul (calculated based on stock concentration) *MALAT1 template (15.54ng/ul)/(0.4/30) = 1165.5ng/ul vs 1485ng/ul (calculated based on stock concentration) *ppillumina_155 (7.02ng/ul)/(0.4/65) = 1140.75ng/ul vs 3267ng/ul (calculated based on stock concentration) ==Probe calculations ppMALAT1== {| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8<\hiddentext> |- align="center" | width="274" height="13" | ppMALAT1 | align="center" width="75" | | align="center" width="155" | | align="center" width="273" | | align="center" width="147" | | align="center" width="238" | | align="center" width="75" | |- align="center" | height="13" | Sample | Conc. (ng/ul) | Volume for 1000ng (ul) | H2O | ppMALAT1 (1434ng/ul) | 10X ampligase Buffer | Total |- align="center" | height="13" | MALAT1 | align="center" | 1165.5 | align="center" | 1 | align="center" | 6 | align="center" | 2 | align="center" | 1 | align="center" | 10 |- align="center" | height="13" | Negavive Control | n/a | n/a | align="center" | 8 | align="center" | 2 | align="center" | 1 | align="center" | 10 |- align="center" | height="13" | Sample | Conc. (ng/ul) | Volume for 10ng (ul) | H2O | ppMALAT1 (10ng/ul) | 10X ampligase Buffer | Total |- align="center" | height="13" | MALAT1 10ng/ul | align="center" | 10 | align="center" | 1 | align="center" | 6 | align="center" | 2 | align="center" | 1 | align="center" | 10 |- align="center" | height="13" | Negavive Control | n/a | n/a | align="center" | 8 | align="center" | 2 | align="center" | 1 | align="center" | 10 |} ==ppMALAT1 Capture Setup== Program stored under Kun/CPG -> 95c 30sec -> cool down to 55C at 0.02C/sec -> 55C 16.5h -> add 3ul ampligase mix (0.5U/ul AmpLigase) -> 55C 5h-> 94C 2min -> add 2ul Exo I/III mix-> 37C 2h -> 94C 2min -> 4C hold. ==PCR Amplification with ISB_CA_AF and ISB_CA_AR_T2 for ppMALAT1== {| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8<\hiddentext> |- style="font-size:12pt" align="center" | width="104" height="15" | Probes | width="144" | ppMALAT1 | width="90" | ppMALAT1 | width="132" | ppMALAT1 | width="90" | ppMALAT1 |- style="font-size:12pt" align="center" | height="15" | Sample | MALAT1 Template (1000ng) | Negative Control | MALAT1 Template (10ng) | Negative Control |- style="font-size:12pt" align="center" | height="15" | Captured Template | align="center" | 5 | align="center" | 5 | align="center" | 5 | align="center" | 5 |- style="font-size:12pt" align="center" | height="15" | 10uM ISB_CA_AF | align="center" | 1 | align="center" | 1 | align="center" | 1 | align="center" | 1 |- style="font-size:12pt" align="center" | height="15" | 10uM ISB_AR_T2 | align="center" | 1 | align="center" | 1 | align="center" | 1 | align="center" | 1 |- style="font-size:12pt" align="center" | height="15" | 2X KAPA SYBR MM | align="center" | 25 | align="center" | 25 | align="center" | 25 | align="center" | 25 |- style="font-size:12pt" align="center" | height="15" | H2O | align="center" | 18 | align="center" | 18 | align="center" | 18 | align="center" | 18 |- style="font-size:12pt" align="center" | height="15" | Total | align="center" | 50 | align="center" | 50 | align="center" | 50 | align="center" | 50 |} stored in #18, #19 Alan Misc. -20C Program (Eppendorf Realplex stored under Alan/ampF6.4_template) 98C 30s -> (98C 10s -> 58C 20s -> 72C 20s)x8 -> (98C 10s -> 72C 20s)x15 -> 72C 3min [[File:01272014.JPG|1000px]] ==Purification & Nanodrop== *Qiaquick: Elute purified amplicons with 30ul EB *10ng:33.9ng/ul * 30ul =1017ng *1000ng:34ng/ul * 30ul = 1020ng ==PAGE Size Verification== [[File:ZhangLab_2 2014-01-27 13hr 47min.jpg|400px]] *size should be at 92bp, but the fact that we see ladder effect (polymerase go around the circle more than once) those bands we see should be the circularized product. I suspect there is actually a band around 90bp but it is to dim to see *Ran denaturing gel to verify [[File:ZhangLab_2 2014-01-27 18hr 41min.jpg|400px]] ==Storage== *Store in Alan Maytag -20C Misc (#30 10ng captured product ~10ul) *Store in Alan Maytag -20C Misc (#31 1000ng captured product ~10ul) *Store in Alan Maytag -20C Misc (#32 10ng amplicon 33.9ng/ul) *Store in Alan Maytag -20C Misc (#33 1000ng amplicon 34ng/ul)
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