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)
Sample | ng/ul | ul | ng/ul | ul | H2O |
ppMALAT1 | 6072 | 0.40 | 20 | 120 | 119.60 |
MALAT1 Template | 1485 | 0.40 | 20 | 30 | 29.6 |
ppillumina_155 | 3267 | 0.40 | 20 | 65 | 64.6 |
10BP | 1000 | 0.40 | 20 | 20 | 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
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
ppMALAT1 | ||||||
Sample | Conc. (ng/ul) | Volume for 1000ng (ul) | H2O | ppMALAT1 (1434ng/ul) | 10X ampligase Buffer | Total |
MALAT1 | 1165.5 | 1 | 6 | 2 | 1 | 10 |
Negavive Control | n/a | n/a | 8 | 2 | 1 | 10 |
Sample | Conc. (ng/ul) | Volume for 10ng (ul) | H2O | ppMALAT1 (10ng/ul) | 10X ampligase Buffer | Total |
MALAT1 10ng/ul | 10 | 1 | 6 | 2 | 1 | 10 |
Negavive Control | n/a | n/a | 8 | 2 | 1 | 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
Probes | ppMALAT1 | ppMALAT1 | ppMALAT1 | ppMALAT1 |
Sample | MALAT1 Template (1000ng) | Negative Control | MALAT1 Template (10ng) | Negative Control |
Captured Template | 5 | 5 | 5 | 5 |
10uM ISB_CA_AF | 1 | 1 | 1 | 1 |
10uM ISB_AR_T2 | 1 | 1 | 1 | 1 |
2X KAPA SYBR MM | 25 | 25 | 25 | 25 |
H2O | 18 | 18 | 18 | 18 |
Total | 50 | 50 | 50 | 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
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
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)