AlanFung:LabNotes/Capturing/2014-1-23: Difference between revisions

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==PAGE Size Verification==
==PAGE Size Verification==
[[File:ZhangLab_2 2014-01-27 13hr 47min.jpg|400px]]
[[File:ZhangLab_2 2014-01-27 13hr 47min.jpg|400px]]
*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.
*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 another gel to verify


==Storage==
==Storage==

Revision as of 02:27, 28 January 2014

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

File:01272014.JPG

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 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 another gel to verify

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)