Matt:LabNotes/2013-7-9

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Revision as of 20:34, 9 July 2013 by >Mzcai (Created page with "===Verify 20 gap Probe Capture=== *Repeat probe capture from [http://genome-tech.ucsd.edu/LabNotes/index.php/Matt:LabNotes/2013-6-28 2013-6-28] due to low capture efficiency o...")
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Verify 20 gap Probe Capture

Synthesis of cDNA from 1ug Human Brain Ref RNA

  • 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 23ul into 1.5ml tube labelled HBRR cDNA 3.11.13)
  • Measured DNA conc with Nandrop: ___
    • ____ sample left in tube

Probe Capture

' Agi26k_20gap CES36k18bp '
Probe Size 12964 13179
gDNA HAPMAP C1 (87ng/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 8.2769x10^8 g/mol (Probe size x (193nt x 325 Da/nt + 79Da)
Amount Probe Required 125 ng (7.44ul) 127 ng (4.47 ul)

0 gap probes from 2013-6-27
20 gap probes from 2013-6-6
cDNA from 2013-6-26

Tube # Probe type Samples Probes (ul) 10X AmpLigase Buffer (ul) Target (ul) H2O (ul) Total
1 0 gap (28.4ng/ul) NTC 4.47 3 0 22.53 30
2 0 gap (28.4ng/ul) gDNA HAPMAP C1 (87ng/ul) 4.47 3 3.45 19.08 30
3 0 gap (28.4ng/ul) cDNA 4.47 3 20 2.53 30
4 20 gap (16.79ng/ul) NTC 7.44 3 0 19.56 30
5 20 gap (16.79ng/ul) gDNA HAPMAP C1 (87ng/ul) 7.44 3 3.45 16.11 30
6 20 gap (16.79ng/ul) cDNA 7.44 3 20 0 30.44

Program

  • 95C 30sec -> cool down to 55 C at 0.02C/sec -> 55 C 20h
  • To 0 gap probes -> add 3ul AmpLigase enzyme mix (0.5U/ul AmpLigase in 1X AmpLigase buffer)
  • To 20 gap probes -> 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.
    • No Exo I/III mix was added to NTC samples because lack of Exo I enzyme