Matt:LabNotes/2017-5-4

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Agi15k_Feb2017 V4 in vitro Capture[edit]

Calculate Probes Needed[edit]

V4[edit]

Probe:target 1000:1 '
Probe size 4998 probes
DNA templet 300 ng
gDNA MW 1.95x10^12 g/mol
gDNA (300ng) 1.5385x10^-19 mol
Probe (1000:1) 1.5385x10^-16 mol
Probe MW (4998, 157nt) 2.387x10^8 g/mol
Amount Probe req'd 36.7 ng


Probes, Target and Ampligase Buffer Mix[edit]

Sample Condition V4 (10.3ng/ul) 12878 DNA (80.3ng/ul) UHRR (1ug/ul) Ampligase Buffer SplintR Buffer Formamide H2O Total
1 Ampligase DNA 3.6 3.8 0 3 0 0 19.6 30
2 Ampligase NTC 3.6 0 0 3 0 0 23.4 30
3 SplintR DNA 3.6 3.8 0 0 3 0 19.6 30
4 SplintR RNA 3.6 0 1 0 3 0 22.4 30
5 SplintR NTC 3.6 0 0 0 3 0 23.4 30
6 SplintR 20% formamide DNA 3.6 3.8 0 0 3 6 13.6 30
7 SplintR 20% formamide RNA 3.6 0 1 0 3 6 16.4 30
8 SplintR 20% formamide NTC 3.6 0 0 0 3 6 17.4 30

Program

  • 1,2,3,6: 95C 30sec -> cool down to 55 C at 0.02C/sec -> 55 C 16h
  • 4,5,7,8: 55C 18h
  • Ampligase: add 3ul AmpLigase enzyme mix (0.5U/ul AmpLigase in 1X AmpLigase buffer)
  • SplintR: add 3ul SplintR enzyme mix (13.5ul SplintR + 2.25ul 10X SplintR Buffer + 6.75ul H2O)
  • Ampligase: -> 55 C 20h -> 94C 2min -> add 2ul Exo I/III mix -> 37C 1h -> 94C 2min -> 4C hold
  • SplintR: -> 37 C 15min -> 94C 10min -> add 2ul Exo I/III mix -> 37C 1h -> 94C 2 min -> 4C hold
  • Zymo columns
    • Elute 10ul
    • Sample 2 was only able to pipette 20ul instead of 30ul of the ligation reaction so theoretically has 33% lower conc after elution from column

AmpLigase enzyme mix[edit]

Components Stock conc. Unit Final conc. Unit Prepare volume 10ul
AmpLigase 5 U/ul 0.5 U/ul 1.00
10x AmpLigase Buffer 10 x 1 x 1.00
H2O 8.00
Total 10.00

Quantify[edit]

  • On 5/18/2017
  1. qPCR all 8 samples 1ul each with triplicates
Components 1X Volume 25X Volume
Captured template 1 0
10uM ISB_CA_AF 0.4 10
10uM ISB_CA_AR.T2 0.4 10
2X KAPA SYBG MM 12.5 312.5
H2O 10.7 267.5
Total 25 600
  • Aliquot 24ul from 24X master mix and add 1ul captured template
 Program
 98C 1min -> (98C 10s -> 52C 20s -> 72C 20s)x30 -> 72C 3min
  • Average Ct values
  DNA RNA NTC
Ampligase 13.7 N/A 27.1
SplintR 12.1 12.1 12.5
SplintR 20% Formamide 19.2 18.9 17.6

Excel data

Conclusion[edit]

  • Accidentally chose 20% Formamide, should have been lower
  • Odd that in 20% Formamide NTC had more captured padlock probes than the EXACT SAME conditions plus DNA or RNA