Noi/NOTES/2012-8-23: Difference between revisions

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* Add 3ul of SLN mix to each
* Add 3ul of SLN mix to each
'''2012.08.25'''<br>
== PCR Amplification with barcoded primers (AmpF6.4Sol and AmpR6.3 IndX XXX) ==
* I tested if the capture works well by performing qPCR of few samples in small volume 25ul
* Sample te be tested: NTC, GL7,GL8 AL-1 and AL3
* Since the total volume of capture reaction is large (55ul), I will add more template in the PCR reaction to the previous experiment (~20% V/V -> 5ul in 25ul reaction)
{| {{table}}
| align="center" style="background:#f0f0f0;"|'''Components'''
| width="50pt" align="center" style="background:#f0f0f0;"|'''1 rxn'''
| width="55pt" align="center" style="background:#f0f0f0;"|'''5.2x rxn mix'''
|-
| Circularized DNA template||5.00||0.00
|-
| 10uM AmpF6.4Sol||0.50||2.60
|-
| 10uM AmpR6.3Sol||0.50||2.60
|-
| 50x SYBG||0.20||1.04
|-
| 2x Phusion MM||12.50||65.00
|-
| H2O||6.30||32.76
|-
| Total||25.00||104.00
|}
'''Program'''
98C 30s -> (98C 10s -> 58C 20s -> 72C 20s)x8 -> (98C 10s -> 72C 20s)x15 -> 72C 3min

Revision as of 23:28, 25 August 2012

Bisulfite conversion

Lab IDs Conc. (ng/ul) Volume for 2000ng LabID ID H2O Total amount (ng) CT conversion reagent (ul) Total volume (ul)
GL-1 295.8 6.76 GL-1 13.24 2000.00 130.00 150.00
GL-2 220.6 9.07 GL-2 10.93 2000.00 130.00 150.00
GL-3 149.7 13.36 GL-3 6.64 2000.00 130.00 150.00
GL-4 172.3 11.61 GL-4 8.39 2000.00 130.00 150.00
GL-5 285.2 7.01 GL-5 12.99 2000.00 130.00 150.00
GL-6 296.6 6.74 GL-6 13.26 2000.00 130.00 150.00
GL-7 320.0 6.25 GL-7 13.75 2000.00 130.00 150.00
GL-8 339.3 5.89 GL-8 14.11 2000.00 130.00 150.00
GL-9 467.5 4.28 GL-9 15.72 2000.00 130.00 150.00
GL-10 431.7 4.63 GL-10 15.37 2000.00 130.00 150.00
GL-11 349.1 5.73 GL-11 14.27 2000.00 130.00 150.00
GL-12 234.5 8.53 GL-12 11.47 2000.00 130.00 150.00
GL-13** 72.2 20.00 GL-13 0.00 1444.80 130.00 150.00
GL-14 137.3 14.56 GL-14 5.44 2000.00 130.00 150.00
GL-15 207.5 9.64 GL-15 10.36 2000.00 130.00 150.00
GL-16 135.5 14.76 GL-16 5.24 2000.00 130.00 150.00
AL-1 226.6 8.83 AL-1 11.17 2000.00 130.00 150.00
AL-2 375.5 5.33 AL-2 14.67 2000.00 130.00 150.00
AL-3 256.8 7.79 AL-3 12.21 2000.00 130.00 150.00
AL-4 142.1 14.08 AL-4 5.92 2000.00 130.00 150.00
AL-5 140.1 14.27 AL-5 5.73 2000.00 130.00 150.00
  • Note: Sample GL-5, GL-7 and GL-8 have lower yield than other samples, so I did bis-cvt in duplicate
Program
1. 98°C for 8 minutes
2. 54°C for 60 minutes
3. 4°C storage for up to 20 hours
  • Elute with 30ul Elution buffer, after Qubit quantification I have ~25ul for BSPP capture

330k BSPP capture set up

Subset ID Adjusted Multiplier(2012_05_22) Actual amount of probe required (probe:target ratio, 100:1) Multiplier F Probe concentration (ng/ul) Volume (ul) Volume for 23rxn (ul) Subset ID
DMR.s1 0.25 2.24 0.56 26.2 0.02 0.49 DMR.s1
DMR.s2 0.35 2.24 0.78 30.1 0.03 0.60 DMR.s2
DMR.s3 1.15 18.90 21.74 21.15 1.03 23.64 DMR.s3
DMR.s4 1.00 18.90 18.90 22.34 0.85 19.46 DMR.s4
DMR.s5 4.83 18.90 91.29 29 3.15 72.40 DMR.s5
DMR.Exp1 1.00 18.90 18.90 31.88 0.59 13.64 DMR.Exp1
DMR.Exp2 1.00 18.90 18.90 24.11 0.78 18.03 DMR.Exp2
DMR.Exp3 1.00 18.90 18.90 28.9 0.65 15.04 DMR.Exp3
1.05uM Oligo suppressor-set1 0.125 2.88 1.05uM Oligo suppressor-set1
1.05uM Oligo suppressor-set2 0.125 2.88 1.05uM Oligo suppressor-set2
  • This means that the maximum volume of bis-cvt gDNA is 42.15ul in total volume 55ul
Components 1rxn (ul) 23 rxn mix (ul)
10X AmpLigase Buffer 5.50 126.50
DMR330k probe mix (ul) 7.10 163.30
105nM Oligo suppressor 0.25 5.75
Total volume (ul) 12.85 295.55


Sample IDs Conc. (ng/ul) Volume for 250ng (ul) Sample IDs H2O Total amount of
bis-cvt gDNA (ng)
Probe mix (ul) 10X AmpLigase Buffer 1.05uM Oligo suppressor
set1 &2 (ul)
Total volume (ul)
GL-1 6.30 39.68 GL-1 2.47 250.00 7.10 5.50 0.25 55.00
GL-2 5.00 42.15 GL-2 0.00 210.75 7.10 5.50 0.25 55.00
GL-3 8.92 28.03 GL-3 14.12 250.00 7.10 5.50 0.25 55.00
GL-4 7.14 35.01 GL-4 7.14 250.00 7.10 5.50 0.25 55.00
GL-5* 5.34 42.15 GL-5* 0.00 225.08 7.10 5.50 0.25 55.00
GL-6## 3.97 42.15 GL-6## 0.00 167.34 7.10 5.50 0.25 55.00
GL-7* 5.30 42.15 GL-7* 0.00 223.40 7.10 5.50 0.25 55.00
GL-8* 5.08 42.15 GL-8* 0.00 214.12 7.10 5.50 0.25 55.00
GL-9 5.51 42.15 GL-9 0.00 232.25 7.10 5.50 0.25 55.00
GL-10 5.31 42.15 GL-10 0.00 223.82 7.10 5.50 0.25 55.00
GL-11 4.99 42.15 GL-11 0.00 210.33 7.10 5.50 0.25 55.00
GL-12 4.97 42.15 GL-12 0.00 209.49 7.10 5.50 0.25 55.00
GL-13 7.11 35.16 GL-13 6.99 250.00 7.10 5.50 0.25 55.00
GL-14 7.27 34.39 GL-14 7.76 250.00 7.10 5.50 0.25 55.00
GL-15 8.01 31.21 GL-15 10.94 250.00 7.10 5.50 0.25 55.00
GL-16 7.25 34.48 GL-16 7.67 250.00 7.10 5.50 0.25 55.00
AL-1 5.06 42.15 AL-1 0.00 213.28 7.10 5.50 0.25 55.00
AL-2 5.00 42.15 AL-2 0.00 210.75 7.10 5.50 0.25 55.00
AL-3 5.22 42.15 AL-3 0.00 220.02 7.10 5.50 0.25 55.00
AL-4 7.16 34.92 AL-4 7.23 250.00 7.10 5.50 0.25 55.00
AL-5 8.40 29.76 AL-5 12.39 250.00 7.10 5.50 0.25 55.00
Program
-> 95c 30sec -> cool down to 55C at 0.02C/sec -> 55C 20h 
-> add 3ul SLN mix (2U/ul AmpliTaq Stoffel fragment; 0.5U/ul AmpLigase; 100uM dNTP) (08.24.2012, ~5pm)
-> 55C 20h-> 94C 2min -> add 2ul Exo I/III mix-> 37C 2h -> 94C 2min -> 4C hold.

SLN mix solution

Components Stock conc. Unit Final conc. Unit Prepare volume72ul
AmpliTaq Stoffel 10 U/ul 2 U/ul 14.40
AmpLigase 5 U/ul 0.5 U/ul 7.20
dNTP 1000 uM 100 uM 7.20
10x AmpLigase Buffer 10 x 1 x 7.20
H2O 36.00
Total 72.00
  • Add 3ul of SLN mix to each

2012.08.25

PCR Amplification with barcoded primers (AmpF6.4Sol and AmpR6.3 IndX XXX)

  • I tested if the capture works well by performing qPCR of few samples in small volume 25ul
  • Sample te be tested: NTC, GL7,GL8 AL-1 and AL3
  • Since the total volume of capture reaction is large (55ul), I will add more template in the PCR reaction to the previous experiment (~20% V/V -> 5ul in 25ul reaction)
Components 1 rxn 5.2x rxn mix
Circularized DNA template 5.00 0.00
10uM AmpF6.4Sol 0.50 2.60
10uM AmpR6.3Sol 0.50 2.60
50x SYBG 0.20 1.04
2x Phusion MM 12.50 65.00
H2O 6.30 32.76
Total 25.00 104.00
Program
98C 30s -> (98C 10s -> 58C 20s -> 72C 20s)x8 -> (98C 10s -> 72C 20s)x15 -> 72C 3min