Matt:LabNotes/2013-7-9: Difference between revisions

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>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...")
 
>Mzcai
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*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 of 20 gap probes and peculiar result for NTC reaction
*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 of 20 gap probes and peculiar result for NTC reaction
*Capture NTC, gDNA, and cDNA with Agi26k_20gap probes and gDNA with [http://genome-tech.ucsd.edu/LabNotes/index.php/CES36k18bp CES36k18bp] as positive control
*Capture NTC, gDNA, and cDNA with Agi26k_20gap probes and gDNA with [http://genome-tech.ucsd.edu/LabNotes/index.php/CES36k18bp CES36k18bp] as positive control
**Alan's previous experiments with CES36k: http://genome-tech.ucsd.edu/LabNotes/index.php/AlanFung:LabNotes/Capturing/2012-5-15
**[http://genome-tech.ucsd.edu/LabNotes/index.php/Capture_Test_of_CES36k18bp Capture Test of CES36k18bp]


====Synthesis of cDNA from 1ug Human Brain Ref RNA====
====Synthesis of cDNA from 1ug Human Brain Ref RNA====
Line 49: Line 49:
{| {{table}}
{| {{table}}
| align="center" style="background:#f0f0f0;"|''''''
| align="center" style="background:#f0f0f0;"|''''''
| align="center" style="background:#f0f0f0;"|'''Agi26k_20gap'''
| align="center" style="background:#f0f0f0;"|'''Agi26k_20gap (16.79ng/ul)'''
| align="center" style="background:#f0f0f0;"|'''CES36k18bp'''
| align="center" style="background:#f0f0f0;"|'''CES36k18bp (75.5ng/ul)'''
| align="center" style="background:#f0f0f0;"|''''''
| align="center" style="background:#f0f0f0;"|''''''
|-
|-
| Probe Size||12964||13179||
| Probe Size||12964||36456||
|-
|-
| gDNA HAPMAP C1 (87ng/ul)||300 ng||300 ng||
| gDNA HAPMAP C1 (87ng/ul)||300 ng||300 ng||
Line 63: Line 63:
| Probe Required (1000:1)||1.5385x10^-16 mol||1.5385x10^-16 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)
| 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)||127 ng (4.47 ul)||
| Amount Probe Required||125 ng (7.44ul)||183 ng (2.42 ul)||
|-
|-
|  
|  
|}
|}


0 gap probes from [http://genome-tech.ucsd.edu/LabNotes/index.php/Matt:LabNotes/2013-6-27 2013-6-27] <br>
20 gap probes from [http://genome-tech.ucsd.edu/LabNotes/index.php/Matt:LabNotes/2013-6-4 2013-6-6] <br>
20 gap probes from [http://genome-tech.ucsd.edu/LabNotes/index.php/Matt:LabNotes/2013-6-4 2013-6-6] <br>
cDNA from [http://genome-tech.ucsd.edu/LabNotes/index.php/Matt:LabNotes/2013-6-26 2013-6-26] <br>


{| {{table}}
{| {{table}}
Line 105: Line 103:
* -> 55 C 20h-> 94C 2min -> add 2ul Exo I/III mix-> 37C 2h -> 94C 2min -> 4C hold.
* -> 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
** No Exo I/III mix was added to NTC samples because lack of Exo I enzyme
====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
|}

Revision as of 20:46, 9 July 2013

Verify 20 gap Probe Capture

  • 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

  • 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 (16.79ng/ul) CES36k18bp (75.5ng/ul) '
Probe Size 12964 36456
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 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 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

AmpLigase enzyme 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
H2O 8.00
Total 10.00

SLN 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
Stoffel 10 U/ul 2 U/ul 2.00
H2O 5.00
Total 10.00