AlanFung:LabNotes/Capturing/2014-1-23: Difference between revisions
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>Alan6017518 |
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(16 intermediate revisions by the same user not shown) | |||
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*Place tray on an orbital shaker for ~5 min. | *Place tray on an orbital shaker for ~5 min. | ||
[[File:ZhangLab_2 2014-01-23 13hr 49min.jpg|400px]] | [[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== | ==ssDNA Qubit to quantify probe and template== | ||
Line 64: | Line 65: | ||
==Probe calculations ppMALAT1== | ==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== | ==ppMALAT1 Capture Setup== | ||
Program stored under Kun/CPG | Program stored under Kun/CPG | ||
Line 69: | Line 136: | ||
-> add 3ul ampligase mix (0.5U/ul AmpLigase) | -> add 3ul ampligase mix (0.5U/ul AmpLigase) | ||
-> 55C 5h-> 94C 2min -> add 2ul Exo I/III mix-> 37C 2h -> 94C 2min -> 4C hold. | -> 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) |
Latest revision as of 02:45, 28 January 2014
Capturing ppMALAT1 template w ppMALT1 ordered form IDT[edit]
TBU GEL to verify probe template size[edit]
- 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[edit]
- 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[edit]
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[edit]
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[edit]
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[edit]
- Qiaquick: Elute purified amplicons with 30ul EB
- 10ng:33.9ng/ul * 30ul =1017ng
- 1000ng:34ng/ul * 30ul = 1020ng
PAGE Size Verification[edit]
File:ZhangLab 2 2014-01-27 13hr 47min.jpg
- 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
Storage[edit]
- 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)