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