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 mNo edit summary |
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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 | ||
** | **[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==== | ||
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{| {{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|| | | 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|| | | 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)|| | | Amount Probe Required||125 ng (7.44ul)||183 ng (2.42 ul)|| | ||
|- | |- | ||
| | | | ||
|} | |} | ||
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> | ||
{| {{table}} | {| {{table}} | ||
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* -> 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 |