AlanFung:LabNotes/Capturing/2014-1-23

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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)