Kun:LabNotes/HiResChrPaint/2012-4-4
Jump to navigation
Jump to search
Processing of oligo pools (STEP 1)
- 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.
Production PCR
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 H2O 44ul 704 =================================== 100ul 95C 30sec -> (95C 3sec -> 55C 30sec-> 72C 20sec) x 15 -> 72C 2min -> 15C hold.
V4 pool for sets #1, #3, #4 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 H2O 44ul 704 =================================== 100ul x 16 95C 30sec -> (95C 3sec -> 55C 30sec-> 72C 20sec) x 16 -> 72C 2min -> 15C hold.