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
File:01272014.JPG
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)