Kun:LabNotes/Haplotyping/2009-2-12: Difference between revisions
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(New page: ==Comparison of MDA using N6, LN6 and N9 primers== *The objective of this experiment is to compare the amplification bias of several variants of MDA protocols: **Control: N6 primer: 30C **...) |
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**LN6 (LNA primer): 30C, 37C, 30C + trehalose | **LN6 (LNA primer): 30C, 37C, 30C + trehalose | ||
**N9: 30C + trehalose | **N9: 30C + trehalose | ||
*The template DNA is GM20431, 30pg/reaction ( | *The template DNA is GM20431, 30pg/reaction (5 copies of diploid genome) | ||
*Two replicates of GM20431 and NTC (20 reactions in total) | *Two replicates of GM20431 and NTC (20 reactions in total) | ||
===Prepare Alkaline Lysis Solution (ALS) and Neutralization Solution (NS)=== | ===Prepare Alkaline Lysis Solution (ALS) and Neutralization Solution (NS)=== | ||
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**Mix 8ul 5M KOH, 10ul 1M DTT, 102ul nuclease free H2O. | **Mix 8ul 5M KOH, 10ul 1M DTT, 102ul nuclease free H2O. | ||
*NS buffer (0.4M HCl, 0.6M Tris.HCl pH 7.5): | *NS buffer (0.4M HCl, 0.6M Tris.HCl pH 7.5): | ||
==Set up MDA reactions== | ==Set up MDA reactions== | ||
*Perform serial dilution of GM20431 gDNA to 30pg/ul. | *Perform serial dilution of GM20431 gDNA to 30pg/ul. | ||
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RepliPhi Phi-29 (100U/ul) 1.0ul 1.0ul | RepliPhi Phi-29 (100U/ul) 1.0ul 1.0ul | ||
*Incubate at 30C for 10h on Opticon4, take one fluorescent read every 6 minutes. | *Incubate at 30C for 10h on Opticon4, take one fluorescent read every 6 minutes. | ||
[[Image:2009-02-12-MDA-N6.png|300px]] [[Image:2009-02-12-MDA-LN6.png|300px]] | |||
[[Image:2009-02-12-MDA-LN6-Tre.png|300px]] [[Image:2009-02-12-MDA-N9-Tre.png|300px]] | |||
[[Image:2009-02-12-MDA-LN6-37C.png|300px]] | |||
==Illumina Infinium Data== | |||
{| {{table}} | |||
| align="center" style="background:#f0f0f0;"|'''GM20431''' | |||
| align="center" style="background:#f0f0f0;"|'''Correct Calls''' | |||
| align="center" style="background:#f0f0f0;"|'''LOH''' | |||
| align="center" style="background:#f0f0f0;"|'''Incorrect Calls''' | |||
| align="center" style="background:#f0f0f0;"|'''No Calls''' | |||
|- | |||
| LN6.GType||186950||56700||56735||249883 | |||
|- | |||
| N6.GType||230134||51403||45395||223336 | |||
|- | |||
| N9+Tre.GType||335118||54811||48774||111565 | |||
|- | |||
| 30pg.GType||303840||52613||47240||146575 | |||
|- | |||
| LN6+Tre.GType||299575||56480||50434||143779 | |||
|- | |||
| | |||
|} | |||
Relative intensity compared with 200ng gDNA | |||
{| {{table}} | |||
| align="center" style="background:#f0f0f0;"|'''Amplicon''' | |||
| align="center" style="background:#f0f0f0;"|'''GM20431 N6.GType''' | |||
| align="center" style="background:#f0f0f0;"|'''GM20431 30 pg.GType''' | |||
| align="center" style="background:#f0f0f0;"|'''GM20431 LN6.GType''' | |||
| align="center" style="background:#f0f0f0;"|'''GM20431 LN6+Tre.GType''' | |||
| align="center" style="background:#f0f0f0;"|'''GM18507 200 ng.GType''' | |||
| align="center" style="background:#f0f0f0;"|'''GM18507 30pg.GType''' | |||
| align="center" style="background:#f0f0f0;"|'''GM20431 N9+Tre.GType''' | |||
|- | |||
| STDEV||1.069||0.975||1.391||0.904||0.208||0.889||0.835 | |||
|- | |||
| Mean||-0.326||-0.124||-0.336||-0.214||0.018||-0.487||-0.160 | |||
|- | |||
| | |||
|} | |||
It looks like the amplicon using the N9 primer + Trehalose has the most number of correct calls. |
Latest revision as of 21:36, 2 April 2009
Comparison of MDA using N6, LN6 and N9 primers[edit]
- The objective of this experiment is to compare the amplification bias of several variants of MDA protocols:
- Control: N6 primer: 30C
- LN6 (LNA primer): 30C, 37C, 30C + trehalose
- N9: 30C + trehalose
- The template DNA is GM20431, 30pg/reaction (5 copies of diploid genome)
- Two replicates of GM20431 and NTC (20 reactions in total)
Prepare Alkaline Lysis Solution (ALS) and Neutralization Solution (NS)[edit]
- ALS: (400mM KOH, 100mM DTT, 10mM EDTA)
- Prepare 5M KOH: weigh one KOH pallet, add nuclease free H2O to make 5M KOH. Make fresh solution every week.
- Mix 8ul 5M KOH, 10ul 1M DTT, 102ul nuclease free H2O.
- NS buffer (0.4M HCl, 0.6M Tris.HCl pH 7.5):
Set up MDA reactions[edit]
- Perform serial dilution of GM20431 gDNA to 30pg/ul.
- Perform UV treatment of ALS and NS buffer 10ul/tube for 5 min;
- Prepare 2X SYBR Green I solution from the 50X stock solution;
- Mix 1ul template with 1.5ul ALS buffer, RT for 3min;
- Transfer to cool block;
- Add 1.5ul NS buffer;
- Add 16ul Master mix:
x13 x9 Tre(-) Tre(+) Trehalose(1.2M) 0ul 5.0ul 10x RepliPhi phi-29 buffer 2.0ul 2.0ul 1mM primer 1.0ul 1.0ul 25mM dNTP 0.8ul 0.8ul 2X SYBR Green I 1.0ul 1.0ul H2O 9.7ul 4.7ul RepliPhi Phi-29 (100U/ul) 1.0ul 1.0ul
- Incubate at 30C for 10h on Opticon4, take one fluorescent read every 6 minutes.
File:2009-02-12-MDA-N6.png File:2009-02-12-MDA-LN6.png File:2009-02-12-MDA-LN6-Tre.png File:2009-02-12-MDA-N9-Tre.png File:2009-02-12-MDA-LN6-37C.png
Illumina Infinium Data[edit]
GM20431 | Correct Calls | LOH | Incorrect Calls | No Calls |
LN6.GType | 186950 | 56700 | 56735 | 249883 |
N6.GType | 230134 | 51403 | 45395 | 223336 |
N9+Tre.GType | 335118 | 54811 | 48774 | 111565 |
30pg.GType | 303840 | 52613 | 47240 | 146575 |
LN6+Tre.GType | 299575 | 56480 | 50434 | 143779 |
Relative intensity compared with 200ng gDNA
Amplicon | GM20431 N6.GType | GM20431 30 pg.GType | GM20431 LN6.GType | GM20431 LN6+Tre.GType | GM18507 200 ng.GType | GM18507 30pg.GType | GM20431 N9+Tre.GType |
STDEV | 1.069 | 0.975 | 1.391 | 0.904 | 0.208 | 0.889 | 0.835 |
Mean | -0.326 | -0.124 | -0.336 | -0.214 | 0.018 | -0.487 | -0.160 |
It looks like the amplicon using the N9 primer + Trehalose has the most number of correct calls.