Dinh 2011/NOTES/2011-8-1
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Barcoding BSPP probes[edit]
0. Oligos re-ordered[edit]
Taqα1_guide_bot: 5'-CAGGCAGATGTTATCGAGGTCCGAC -3' (Tm ~ 68C) Taqα1_insert_guide_bot: 5'-GTCGGACCTCGTGAGAGTGGTGTANNNNNNNGATCTCGGTGGTCGCCGTATCATTATAACATCTGCCTG - 3' (Tm ~ 81C) Insert = 44 residues
APV6_Splint: 5'-TACACACTACCCGTGGGGTCATATCGGTCA-3' (Tm ~ 73C)
1. Production of probes[edit]
- Make master mix of all 1st round amplicon probes subset with multiplex E ratios (see Noi's notes)
SUBSET Vol(ul) S1 (diluted to 0.1x) 0.33 S2 (diluted to 0.1x) 0.48 S3 1.32 S4 5.67 S5 27.39 Exp1 1.33 Exp2 1.05 Exp3 1.51 -------------------------
- PCR reaction of probes with phosphorylated primer at common flanking sequence
Production PCR: 1x 32x 10nM 1st round amplicon 0.2ul 6.4ul 2x Kapa SYBR qPCR MM 50ul 1600ul 100uM pAP1V61U 0.4ul 12.8ul 100uM AP2V6 0.4ul 12.8ul H2O 49ul 1568ul ---------------------------------------------- 100ul x 32 ---------------------------------------------- 95C 30s -> (95C 3s -> 55C 30s -> 60C 20s) x 14 cycles -> 60C 2min -> 15C hold Ethanol precipitation, re-purified with Qiaquick columns elute with EB. Yield (Nanodrop): 74ng/ul * 120 ul + 78ng/ul * 120 = 18.240 ug
2. Circularization with circle-helper and Ampligase[edit]
- Ampligase to anneal 5' to 3' ends with APV6_Splint (1:1 ratio)
Circularization reaction: 1 ug of dsDNA probes (150 nt, MW = 91,268 g/moles) Probes (1.09x10^-11 moles in 1 ug) APV6_Splint (1.09x10^-11 moles = 100x10^-12 moles/ul*0.109ul) Splint : Use 0.109ul 100uM APV6_Splint per 1 ug of probes (1:1 ratio). Ampligase (Epicentre) : Use 1 ul of Ampligase Ampligase buffer : Use 1x
Reaction: 1 ug amplified probes (74ng/ul) 13.5 ul APV6_Splint (5uM) 2.2 ul dilute APV6_Splint (5 ul + 95 ul ddH20) Ampligase 1.0 ul Ampligase buffer 2.0 ul ddH2O 1.3 ul ----------------------------------------- 20.0 ul total
Program: 95C 30S -> 94.8C for 30s, -0.2C per cycle x 148 cycles -> (65C 10min -> 94C 30s)x10 -> 65C forever -> 37C 30 s -> Add 1 ul ExoI and ExoIII each -> 37C 45 min -> 4C hold
- Exonuclease I/III to remove all linear DNA
Purified with columns, elute with 30ul EB. Nanodrop: 1.6ng/ul, 2.43 OD
Repeat circularization due to low yield
Reaction: 1 ug amplified probes (74ng/ul) 13.7 ul APV6_Splint (5uM) 3.3 ul 3:2 ratio Final conc = 825nM => Tm = 73.14C Ampligase 1.0 ul Ampligase buffer 2.0 ul ----------------------------------------- 20.0 ul total
Program A and B: 95C 30S -> [94.8C for 30s, -0.2C per cycle x 148 cycles -> 65C hold -> add 1ul Ampligase]x accidentally cycled here for total 2x ->(65C 10min -> 94C 30s)x11 -> 65C forever -> 37C 1 min -> Add 1 ul ExoI and ExoIII each -> 37C 45 min -> 90C 10 min 4C hold
Program C: 95C 30S -> 94.8C for 30s, -0.2C per cycle x 148 cycles -> 65C hold -> add 1ul Ampligase ->(65C 10min -> 94C 30s)x11 -> 65C forever -> 37C 1 min -> Add 1 ul ExoI and ExoIII each -> 37C 45 min -> 90C 10 min-> 4C hold
Program D: (94C 30s -> 65C 2 min)x40 -> 65C forever -> 37C 1 min -> Add 1 ul ExoI and ExoIII each -> 37C 1 hr -> 90C 10 min -> 4C hold
- Quantify amount of circularized probes using TBU gel
File:ZhangLab 2 2011-08-10 DMR330K-E circularization.png A - Low mass ladder (0.6ul) B - Ligation product from program C (can see the circularized probes band) C - Ligation product from program D (incubation with Ampligase at high temperature have exonucleas activity ?) D - Linear dsDNA probes E - 10 bp ladder
- Adding ampligase after gradual lowering of temperature from 95C helps, allows for longer incubation at 65C?
- Try titrating the amount of APV6_Splint to be added
Repeat circularization with titrating splint amount
Reaction: 1 ug amplified probes (78ng/ul) 12.8 ul APV6_Splint (1uM) probes to splint 1:1 = 11 ul 1:2 = ul 1:4 = 22 ul Ampligase buffer 2.7 ul H2O 1:1 = 0.5 ul 1:2 = 6.0 ul 1:4 = 8.75 ul ----------------------------------------- 27.0 ul total Program: 95C 30S -> 94.8C for 30s, -0.2C per cycle x 199 cycles -> 55C hold -> add 1ul Ampligase @ 20 min -> 55C 2 hr (remove 5ul for gel) -> 37C 1 min -> Add 1 ul ExoI/ExoIII mix -> 37C 1 hr -> 90C 10 min-> 4C hold
File:TitratingCircularization.jpg
Mix products from all tubes. Purified with 1.8x AMPure beads, elute with water. Nanodrop: 1.6ng/ul
3. Annealing of guide oligo and digestion[edit]
- Quantify amount of circularized ssDNA probes using gel
- Use 5:1 ratios of guide oligos
Circularized probes: 1 ug of ssDNA circular probes (150 nt, MW = 45,634 g/moles) = 2.191x10^-11 moles
TaqαI_Guide: 5 * 2.191x10^-11 moles / 100x10^-12 moles/ul = 1.0955 ul -Use 1.1 ul 100uM TaqαI guide oligo per 1 ug of circle ssDNA probes
TaqαI : 1 unit cuts 121 sites in 1 ug Lambda DNA (48,502bp, MW = 2.946x10^7g/mol) in 1 hour at 65C Unit per sites*moles = 1 unit * 121^-1 sites^-1 * 2.946x10^13 mol^-1 = 2.946x10^11 -Use 2.946x10^-11 sites*moles x2.191x10^-11 = 3.523 units per 1 ug of circle ssDNA probes
Reaction: 20 ul Circled probes 0.64 ul TaqαI_Guide (0.5uM) 1 ul TaqαI (20U/ul) 4 ul NEB Buffer 4 (10x) 0.4 ul BSA (100x) --------------------- 40ul total
Program: 65C 20min -> (Add enzyme) 65C for 1 hr -> 80C 20 min -> 4C hold Purify with 1 column, elute in water Nanodrop: too low
Gel: File:Taq1digest.jpg
4. Annealing of guide oligo, fill in with Stoffel, and Ampligase[edit]
- Set up as in capture reaction but with higher annealing temperature
- Use 2:1 ratio of guide oligos
TaqαI digested ssDNA probes : 1ug 1 ug of ssDNA probes (150 nt, MW = 45,634 g/moles) = 2.191x10^-11 moles
TaqαI_RandBar_guide : - Use 0.109 ul of 100uM Insert_guide oligo per 1 ug of digested probes Stoffel (10U/ul): One unit per 10nmoles dNTPs to incorporate per 30 min in 10 min incubation at 74C 0.0219nmolesx44nt = 0.9636nmoles dNTPs to incorporate Reaction will cycle to 74C for a total of 30 min Assumes One unit per 40nmoles, thus ~0.5 Units Stoffel per 1 ug of digested ssDNA probes. dNTP (10mM = 10nmoles/ul): Optimal is 10x the amount required for 100% of targets. 0.0219nmolesx44nt = 0.9636 nmoles dNTPs to incorporate 100*0.9636nmoles/10nmoles/ul = 9.636ul - For 100:1 guide to probes ratio, use 10 ul of 10mM dNTP per 1 ug of digested ssDNA probes
Ampligase (5U/ul): One unit per 50% of cos site of 1 ug lambda DNA in 1 minute at 45C - Use 1 unit Ampligase buffer (10x): Use 1x
Reaction Mix: SLN for ~ 5 ng of digested product Use 0.5nmoles of dNTP = (1nmoles/ul)x 0.5 ul 0.5 ul dNTP (1 mM) 1.0 ul Stoffel (10U/ul) 0.5 ul Ampligase (5U/ul) 0.5 ul Ampligase buffer (10x) 2.5 ul H2O --------------------------- 5.0 ul total Program: Program C: 95C 30S -> 94.8C for 30s, -0.2C per cycle x 184 cycles -> 58C hold -> add SLN ->(58C 10min -> 94C 30s)x11 -> 58C forever -> 37C 1 min -> Add 1 ul ExoI and ExoIII each -> 37C 45 min -> 90C 10 min-> 4C hold
5. PCR Amplification of circled probes with common sequence primers[edit]
- PCR reaction of probes with primer at common flanking sequence
Production PCR: 1x 32x circled barcoded probes ul ul 2x Kapa SYBR qPCR MM ul ul 100uM pAP1V61U ul ul 100uM AP2V6 ul ul H2O ul ul ---------------------------------------------- 100ul x 32 ---------------------------------------------- 95C 30s -> (95C 3s -> 55C 30s -> 60C 20s) x 15 cycles -> 60C 2min -> 15C hold Ethanol precipitation, re-purified with Qiaquick columns elute with EB. Yield (Nanodrop):
6. Probes preparation - adapters removal[edit]
7. Capture using barcoded probes[edit]
8. Amplification with AmpR6.3IndXX and AmpF6.4 primers[edit]
9. Set up new recipe on GAIIx[edit]
- Run sequencing to sequence 4 reads using 4 set of reading primers