Matt:LabNotes/2013-7-9

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Revision as of 23:51, 10 July 2013 by >Mzcai (→‎SLN mix)
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Verify 20 gap Probe Capture[edit]

  • Repeat probe capture from 2013-6-28 due to low capture efficiency of 20 gap probes and peculiar result for NTC reaction
  • Capture NTC, gDNA, and cDNA with Agi26k_20gap probes and gDNA with CES36k18bp as positive control

Synthesis of cDNA from 1ug Human Brain Ref RNA[edit]

  • Followed NEB E6300S protocol: [1]
  • Used 1 ul of Human Brain Reference RNA at concentration 1ug/ul for cDNA synthesis
  • Mixed components in 0.2 ml microfuge tube labelled HBRR 1ug cDNA
Components Volume
RNA 1 ul (1 ug)
d(T)23VN (50 µM) 2 ul
H2O 5 ul
Total 8 ul
  • Denatured RNA for 5 min at 70C and then put on ice
  • Added the following to tube
Components Volume
M-MuLV Reaction Mix 10 ul
M-MuLV Enzyme Mix 2 ul
Total 20 ul
  • Incubated for one hour at 42C
  • Inactivated enzyme for 5 min at 80C
  • Followed NEB E6111S protocol: [2]
  • Added 48 ul H20
  • Added 8ul 10x Second strand synthesis reaction buffer
  • Added 4ul Second strand synthesis enzyme mix
  • Mixed by pipetting
  • Incubated for 2.5 hours at 16C
  • Purified using Qiagen minelute column (eluted 21ul)
  • Measured DNA conc with Nandrop: 47ng/ul
    • 19.5 ul (916.5 ng) sample left in tube

Probe Capture[edit]

' Agi26k_20gap (16.79ng/ul) CES36k18bp (75.5ng/ul) '
Probe Size 12964 36456
gDNA 12878 (100ng/ul) 300 ng 300 ng
gDNA MW 1.95x10^12 g/mol 1.95x10^12 g/mol
gDNA 1.5385x10^-19 mol 1.5385x10^-19 mol
Probe Required (1000:1) 1.5385x10^-16 mol 1.5385x10^-16 mol
Probe MW 8.1419x10^8 g/mol 1.1877x10^9 g/mol (Probe size x (Probe length x 325 Da/nt + 79Da)
Amount Probe Required 125 ng (7.44ul) 183 ng (2.42 ul)

20 gap probes from 2013-6-6

Tube # Probe Type Sample Probe (ul) 10X Ampligase Buffer Target (ul) H2O Total
1 Agi26k_20gap NTC 7.44 3 0 19.56 30
2 Agi26k_20gap gDNA 12878 7.44 3 3 16.56 30
3 Agi26k_20gap cDNA 7.44 3 19 0.56 30
4 CES36k18bp gDNA 12878 2.42 3 3 21.58 30

Program

  • 95C 30sec -> cool down to 55 C at 0.02C/sec -> 55 C 20h
  • -> add 2ul SLN mix (2U/ul AmpliTaq Stoffel fragment; 0.5U/ul AmpLigase; 100uM dNTP mix)
  • -> 55 C 20h-> 94C 2min -> add 2ul Exo I/III mix-> 37C 2h -> 94C 2min -> 4C hold.

SLN mix[edit]

  • Noi made the mix (NEED TO UPDATE THIS SECTION WHEN DETAILS ARE UP ON NOI'S WIKI)
    • Noi heat activated the Taq polymerase before adding to the mix
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
dNTP Mix 1 mM 100 uM 1.00
Titanium Taq DNA 10 U/ul 2 U/ul 2.00
H2O 5.00
Total 10.00