Kun:LabNotes/HiResChrPaint/2012-4-4
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Processing of oligo pools (STEP 1)[edit]
- Received the oligo pools from Mycorarray in four tubes. These are "ssDNA from emulsion PCR pool". Resuspend each pool in 500ul nuclease free H2O.
- Take 50ul of each oligo pool, dilute to 20nM. These will be the working tubes.
5431_ssDNA 80k_set_4 ~2.8ug 85pmole 170nM 50ul+374ul H2O => 20nM 5432_ssDNA 80k_set_1 ~1.8ug 55pmole 109nM 50ul+223ul H2O => 20nM 5433_ssDNA 80k_set_2 ~1.7ug 52pmole 103nM 50ul+208ul H2O => 20nM 5435_ssDNA 80k_set_3 ~2.8ug 85pmole 170nM 50ul+374ul H2O => 20nM
- There is probably no need to do expansion PCR since the oligo pools were already amplified by emulsion PCR.
Confirmation PCR[edit]
V6 pool Each 20nM oligo pool 0.4ul 2x Kapa SYBG qPCR Master Mix 50ul 100uM pAP1V61U 0.4ul 100uM AP2V6 0.4ul 2mM aa-dUTP 3ul H2O 41ul =================================== 100ul 95C 30sec -> (95C 3sec -> 55C 30sec-> 72C 20sec) x 15 -> 72C 2min -> 15C hold.
V4 pool Each 20nM oligo pool 0.4ul 2x Kapa SYBG qPCR Master Mix 50ul 100uM pAP1V41U 0.4ul 100uM AP2V4 0.4ul 2mM aa-dUTP 3ul H2O 41ul =================================== 100ul 95C 30sec -> (95C 3sec -> 55C 30sec-> 72C 20sec) x 16 -> 72C 2min -> 15C hold. Check the size distribution of the amplicons with 6% TBE gels.
File:4.4.12 oligopaint 1-4.jpg File:4.4.12 oligopaint 5-8.jpg
Production PCR[edit]
V6 pool for all four sets Each x16 20nM oligo pool 0.2ul 3.2 2x Kapa SYBG qPCR Master Mix 50ul 800 100uM pAP1V61U 0.4ul 6.4 100uM AP2V6 0.4ul 6.4 2mM aa-dUTP 3ul 48 H2O 41ul 656 =================================== 100ul 95C 30sec -> (95C 3sec -> 55C 30sec-> 72C 20sec) x 15 -> 72C 2min -> 15C hold.
V4 pool for all four sets. Each x16 20nM oligo pool 0.2ul 3.2 2x Kapa SYBG qPCR Master Mix 50ul 800 100uM pAP1V41U 0.4ul 6.4 100uM AP2V4 0.4ul 6.4 2mM aa-dUTP 3ul 48 H2O 41ul 656 =================================== 100ul x 16 95C 30sec -> (95C 3sec -> 55C 30sec-> 72C 20sec) x 16 -> 72C 2min -> 15C hold. Check the size distribution of the amplicons with 6% TBE gels. Ethanol precipitation. Re-purified each amplicon with two Qiaquick columns, eluted with 40ul EB. Measure concentration.
Dpn II digestion[edit]
Lambda exo digestion[edit]
Coupling of Alexa fluorophores with ss-DNA probes[edit]
Adaptor removal[edit]
- Dpn II digestion.
ds-oligo pools 75ul 10X Dpn II buffer 10ul Dpn II (10U/ul) 8ul (might need to adjust the amount of enzyme based on the yield of PCR) H2O 7ul 37C 30min -> 65C 20min -> Qiaquick column purification
- Lambda exo digestion.
Dpn II digested oligo pool 40ul 10X Lambda Exo buffer 10ul Lambda Exo (10U/ul) 10ul H2O 40ul 37C 1h -> 75C 10min -> Qiaquick column purification
- Proceed with AlexaFluor coupling.