Matt:LabNotes/2013-6-28: Difference between revisions

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'''Program'''<br>
* 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
Continued on: http://genome-tech.ucsd.edu/LabNotes/index.php/Matt:LabNotes/2013-7-1
====AmpLigase enzyme mix====
{| class="wikitable" style="text-align:center;{{table}} border = 1
| align="center" style="background:#f0f0f0;"|'''Components'''
| align="center" style="background:#f0f0f0;"|'''Stock conc.'''
| align="center" style="background:#f0f0f0;"|'''Unit'''
| align="center" style="background:#f0f0f0;"|'''Final conc.'''
| align="center" style="background:#f0f0f0;"|'''Unit'''
| align="center" style="background:#f0f0f0;"|'''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
|}
====SLN mix====
{| class="wikitable" style="text-align:center;{{table}} border = 1
| align="center" style="background:#f0f0f0;"|'''Components'''
| align="center" style="background:#f0f0f0;"|'''Stock conc.'''
| align="center" style="background:#f0f0f0;"|'''Unit'''
| align="center" style="background:#f0f0f0;"|'''Final conc.'''
| align="center" style="background:#f0f0f0;"|'''Unit'''
| align="center" style="background:#f0f0f0;"|'''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
|-
| Stoffel||10||U/ul||2||U/ul||2.00
|-
| H2O||||||||||5.00
|-
| Total||||||||||10.00
|}
|}

Latest revision as of 19:20, 1 July 2013

Probe Capture[edit]

' 20 gap 0 gap '
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

Continued on: http://genome-tech.ucsd.edu/LabNotes/index.php/Matt:LabNotes/2013-7-1

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

SLN 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
dNTP Mix 1 mM 100 uM 1.00
Stoffel 10 U/ul 2 U/ul 2.00
H2O 5.00
Total 10.00