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

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>Alan6017518
Line 68: Line 68:
  -> 95c 30sec -> cool down to 55C at 0.02C/sec -> 55C 16.5h  
  -> 95c 30sec -> cool down to 55C at 0.02C/sec -> 55C 16.5h  
  -> add 3ul ampligase mix (0.5U/ul AmpLigase)
  -> add 3ul ampligase mix (0.5U/ul AmpLigase)
  -> 55C 20h-> 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.

Revision as of 18:38, 27 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

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 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.