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Kun:LabNotes/Haplotyping/2009-2-12
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==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 **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)=== *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== *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. [[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.
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