Dinh 2011/NOTES/2011-7-5
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Protocol Outline and Considerations for barcoding probes[edit]
0. Oligos ordered[edit]
- TaqαI_Guide: 5- GTC GGA CCT CGA TAA CAT CTG CCT G -3 (Tm = 60.8C)
- TaqαI_RandBar_Guide: 5- CAG GCA GAT GTT ATA ATG ATA CGG CGA CCA CCG AGA TCN NNN NNN TAC ACC ACT CTC ACG AGG TCC GAC -3 (Tm=71C)
- APV6_splint: 5- CAG GAT ACA CAC TAC CCG TGA CAG TGA CCG ATA TGA CCC -3 (Tm = 66.7C)
1. Production of probes[edit]
- 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 15 cycles -> 60C 2min -> 15C hold Ethanol precipitation, re-purified with Qiaquick columns elute with EB. Yield (Nanodrop):
2. Circularization with circle-helper and Ampligase[edit]
- Ampligase to anneal 5' to 3' ends with APV6_Splint (1:1 ratio)
Sequence to be ligated: 5'-AACAGTGACCGATATGACCC*CACGGGTAGTGTGTATCCTG-3' APV6_Splint: 5'-TACACACTACCCGTG*GGGTCATATCGGTCA-3'
Circularization reaction: 1 ug of dbDNA probes (150 nt, MW = 45,634g/moles) Probes (2.19x10^-11 moles in 1 ug) APV6_Splint (2.19x10^-11 moles = 100x10^-12 moles/ul*0.219ul) Splint : Use 0.219ul 100uM APV6_Splint per 1 ug of probes. Ampligase (Epicentre) : Use 1 ul of Ampligase Ampligase buffer : Use 1x 94C 3min -> (94C 30s -> 65C 10min)x10 -> 4C
- Exonuclease I/III to remove all linear DNA
Add 1 ul of ExonI and 1 ul ExoIII 37C for 30 min Purify with columns, elute with EB.
- Quantify amount of circularized probes using TBU gel
3. Annealing of guide oligo and digestion[edit]
- Quantify amount of circularized probes using gel
- Add 1:1 ratios of guide oligos
Circularized probes: --- TaqαI_Guide : Use 0.219ul 100uM TaqαI guide oligo per 1 ug of circle probes TaqαI : Use 1 ul per 1 ug of circle probes 95C 10 min -> gradient to 65C -> 65C 20min -> (Add enzyme) 65C for 2 hr Purify with columns, elute in EB, and quantify with Nanodrop
4. Annealing of guide oligo, fill in with Stoffel, and Ampligase[edit]
- Set up as in capture reaction but with higher annealing temperature
TaqαI digested probes : --- TaqαI_RandBar_guide : Use 0.219 ul of 100uM Insert_guide oligo per 1 ug of digested probes Ampligase : Use 1 ul Ampligase buffer : Use 1x 94C 3min -> 60C 4 hr -> Add 2 ul SLN -> 60C 4 hr -> 37C 1 min -> Add 2ul ExoI/ExoIII -> 37C 1 hr -> 90C for 10 min -> 4C hold Purify with TBU gel size selection and quantification with TBU-gel
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
To get 70% circularization in paper published[edit]
[1] "Rolling circle amplification under topological constraints"
Circularizable DNA - CTGGAGGAGATTTTGTGGTATCGATTCGTCTCTTAGAGGAAGCTA
- I don't know why their splint has a non-complementary CGATC tail
Splint - GATCG_TCTCCTCCAGTAGCTTCCTC rc-Splint - GAGGAAGCTACTGGAGGAGA_CGATC