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
- 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
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
- size should be at 92bp, but since circularized product move slower in GEL, and the fact that we see ladder effect (polymerase go around the circle more than once) those bands we see should be the circularized product.
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)