Noi/NOTES/2014-5-23: Difference between revisions

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* I include sample C1, A1, and A2 in capture reaction, but not include GP1V4. This is  may not necessary, but I want to make sure that there is no any background from the template itself.
* I include sample C1, A1, and A2 in capture reaction, but not include GP1V4. This is  may not necessary, but I want to make sure that there is no any background from the template itself.
* For cancer samples, I performed bisulfite conversion 6tubes of C1 and 3tubes of C2 samples (600ng gDNA for each tube). I used bisulfite treated DNA from different tubes for each reaction. There might me some variations of DNA amount in each reaction based on accuracy of DNA concentration quantification or pipetting.
* For cancer samples, I performed bisulfite conversion 6tubes of C1 and 3tubes of C2 samples (600ng gDNA for each tube). I used bisulfite treated DNA from different tubes for each reaction. There might me some variations of DNA amount in each reaction based on accuracy of DNA concentration quantification or pipetting.
== Bisulfite conversion ==
* I did bisulfite converssion by usingEZ DNA Methylation-Lightning™ Kit -Zymo and followed the protocol of the kit
* Follow the instruction of the kit
  '''Program'''
1. 98°C for 8 minutes
2. 54°C for 60 minutes
3. 4°C hold
* Elute with 16ul elution buffer. Total volume left after elution ~15ul
* Took 2ul for Qubit ssDNA assay
:{| {{table}} class = wikitable
| align="center" style="background:#f0f0f0;"|'''Sample'''
| align="center" style="background:#f0f0f0;"|'''Concentration in the Qubit'''
| align="center" style="background:#f0f0f0;"|''''''
| align="center" style="background:#f0f0f0;"|'''uL used'''
| align="center" style="background:#f0f0f0;"|'''Dilution'''
| align="center" style="background:#f0f0f0;"|'''Sample Concentration'''
| align="center" style="background:#f0f0f0;"|''''''
| align="center" style="background:#f0f0f0;"|'''Yield in 15ul (ng)'''
| align="center" style="background:#f0f0f0;"|'''% Recovery'''
|-
| CANC1-1||219||ng/mL||2||100||21.9||ug/mL||328.50||54.75
|-
| CANC1-2||204||ng/mL||2||100||20.4||ug/mL||306.00||51.00
|-
| CANC1-3||231||ng/mL||2||100||23.1||ug/mL||346.50||57.75
|-
| CANC1-4||209||ng/mL||2||100||20.9||ug/mL||313.50||52.25
|-
| CANC1-5||226||ng/mL||2||100||22.6||ug/mL||339.00||56.50
|-
| CANC1-6||227||ng/mL||2||100||22.7||ug/mL||340.50||56.75
|-
| CANC2-1||207||ng/mL||2||100||20.7||ug/mL||310.50||51.75
|-
| CANC2-2||195||ng/mL||2||200||19.5||ug/mL||292.50||48.75
|-
| CANC2-3||209||ng/mL||2||100||20.9||ug/mL||313.50||52.25
|}
== BSPP capture set up ==
* BSC_A1 conc. = 120ng/ul
* BSC_A2 conc. = 100ng/ul
* GP1V4 conc. (prep 2014-05-20) = 8.37ng/ul
==== Probe & AmpLigase Buffer mix ====
===== Mix I. Amligase buffer and GP1V4 =====
:{| {{table}} class = wikitable
| align="center" style="background:#f0f0f0;"|'''Components'''
| align="center" style="background:#f0f0f0;"|'''1 rxn'''
| align="center" style="background:#f0f0f0;"|'''18 rxn mix'''
|-
| GP1V4 (8.37ng/ul)||1.55||27.96
|-
| 10X AmpLig Buffer||2.00||36.00
|-
| H2O||5.00||90.00
|-
| Total||8.55||
|}
===== Mix II. only Amligase buffer =====
:{| {{table}} class = wikitable
| align="center" style="background:#f0f0f0;"|'''Components'''
| align="center" style="background:#f0f0f0;"|'''1 rxn'''
| align="center" style="background:#f0f0f0;"|'''11 rxn mix'''
|-
| 10X AmpLig Buffer||2.00||22.00
|-
| H2O||5.00||55.00
|-
| Total||7.00||
|}
{| {{table}} class = wikitable
| align="center" style="background:blue;"|'''Stoffel'''
| align="center" style="width:50px; background:#f0f0f0;"|'''C1-S'''
| align="center" style="width:50px; background:#f0f0f0;"|'''C2-S'''
| align="center" style="width:50px; background:#f0f0f0;"|'''A1-S'''
| align="center" style="width:50px; background:#f0f0f0;"|'''A2-S'''
| align="center" style="width:50px; background:#f0f0f0;"|'''NTC'''
| align="center" style="width:50px; background:#f0f0f0;"|'''C1 no GP1V4'''
| align="center" style="width:50px; background:#f0f0f0;"|'''A1 no GP1V4'''
| align="center" style="width:50px; background:#f0f0f0;"|'''A2 no GP1V4'''
|-
| Conc. (ng/ul)||21.90||20.70||120.00||100.00||||20.40||120.00||100.00
|-
| Vol. for 200ng||9.13||9.66||1.67||2.00||0.00||9.80||1.67||2.00
|-
| GP1V4 (8.37ng/ul)||1.55||1.55||1.55||1.55||1.55||0.00||0.00||0.00
|-
| H2O||2.31||1.78||9.78||9.45||11.45||3.20||11.33||11.00
|-
| 10X Ampligase buffer||2.00||2.00||2.00||2.00||2.00||2.00||2.00||2.00
|-
| H2O in Mix||5.00||5.00||5.00||5.00||5.00||5.00||5.00||5.00
|-
| Total||20.00||20.00||20.00||20.00||20.00||20.00||20.00||20.00
|}
{| {{table}} class = wikitable
| align="center" style="background:red;"|'''Hemo Klentaq'''
| align="center" style="width:50px; background:#f0f0f0;"|'''C1-S'''
| align="center" style="width:50px; background:#f0f0f0;"|'''C2-S'''
| align="center" style="width:50px; background:#f0f0f0;"|'''A1-S'''
| align="center" style="width:50px; background:#f0f0f0;"|'''A2-S'''
| align="center" style="width:50px; background:#f0f0f0;"|'''NTC'''
| align="center" style="width:50px; background:#f0f0f0;"|'''C1 no GP1V4'''
| align="center" style="width:50px; background:#f0f0f0;"|'''A1 no GP1V4'''
| align="center" style="width:50px; background:#f0f0f0;"|'''A2 no GP1V4'''
|-
| Conc. (ng/ul)||23.10||19.50||120.00||100.00||||20.90||120.00||100.00
|-
| Vol. for 200ng||8.66||10.26||1.67||2.00||0.00||9.57||1.67||2.00
|-
| GP1V4 (8.37ng/ul)||1.55||1.55||1.55||1.55||1.55||0.00||0.00||0.00
|-
| H2O||2.79||1.19||9.78||9.45||11.45||3.43||11.33||11.00
|-
| 10X Ampligase buffer||2.00||2.00||2.00||2.00||2.00||2.00||2.00||2.00
|-
| H2O in Mix||5.00||5.00||5.00||5.00||5.00||5.00||5.00||5.00
|-
| Total||20.00||20.00||20.00||20.00||20.00||20.00||20.00||20.00
|}
{| {{table}} class =wikitable
| align="center" style="background:dimgray;"|'''ILMN-Pol'''
| align="center" style=width:50px; "background:#f0f0f0;"|'''C1-S'''
| align="center" style=width:50px; "background:#f0f0f0;"|'''C2-S'''
| align="center" style=width:50px; "background:#f0f0f0;"|'''A1-S'''
| align="center" style=width:50px; "background:#f0f0f0;"|'''A2-S'''
| align="center" style=width:50px; "background:#f0f0f0;"|'''NTC'''
| align="center" style=width:50px; "background:#f0f0f0;"|'''C1 no GP1V4'''
| align="center" style=width:50px; "background:#f0f0f0;"|'''A1 no GP1V4'''
| align="center" style=width:50px; "background:#f0f0f0;"|'''A2 no GP1V4'''
|-
| Conc. (ng/ul)||22.60||20.90||120.00||100.00||||22.70||120.00||100.00
|-
| Vol. for 200ng||8.85||9.57||1.67||2.00||0.00||8.81||1.67||2.00
|-
| GP1V4 (8.37ng/ul)||1.55||1.55||1.55||1.55||1.55||0.00||0.00||0.00
|-
| H2O||2.60||1.88||9.78||9.45||11.45||4.19||11.33||11.00
|-
| 10X Ampligase buffer||2.00||2.00||2.00||2.00||2.00||2.00||2.00||2.00
|-
| H2O in Mix||5.00||5.00||5.00||5.00||5.00||5.00||5.00||5.00
|-
| Total||20.00||20.00||20.00||20.00||20.00||20.00||20.00||20.00
|}
'''Program'''
-> 95c 30sec -> cool down to 55C at 0.02C/sec -> 55C 20h
-> add 2ul SLN mix (2U/ul '''Polymerase''' (NEB); 0.5U/ul AmpLigase; 100uM dNTP)
-> 55C 20h-> 94C 2min -> add 2ul Exo I/III mix-> 37C 2h -> 94C 2min -> 4C hold.
* For Illumina's polymerase I used 1ul for each sample as recommended by Xuyu
==== SLN mix solution ====

Revision as of 21:43, 26 May 2014

BSPP capture of amplified DNA from Illumina using different 3 enzymes for polymerization

[Link to calendar]

  • From previous experiment on 2014-05-13 [[1]], the results were inconclusive, and I lacked positive control. Dr. Zhang suggested to repeat experiment on few samples from Illumina and include cancer sample DNA as control.
  • I need to prepare more GP1V4 BSPP.

Sample list and experimental design

  • Cancer sample #1 or C1 (BXPC3)
  • Cancer sample #2 or C2 (U87MG)
  • Illumina amplified DNA, A1
  • Illumina amplifed DNA, A2
  • Capture with 500:1 GP1V4:target ratio (total amount in each reaction ~13ng)
  • Bisulfite treated DNA/amplified DNA amount 200ng
  • Add different enzyme in SLN mix,
    • Stoffel (blue color)
    • Hemo KlenTaq (red color)
    • Illumina Polymerase (black color)
  • I include sample C1, A1, and A2 in capture reaction, but not include GP1V4. This is may not necessary, but I want to make sure that there is no any background from the template itself.
  • For cancer samples, I performed bisulfite conversion 6tubes of C1 and 3tubes of C2 samples (600ng gDNA for each tube). I used bisulfite treated DNA from different tubes for each reaction. There might me some variations of DNA amount in each reaction based on accuracy of DNA concentration quantification or pipetting.

Bisulfite conversion

  • I did bisulfite converssion by usingEZ DNA Methylation-Lightning™ Kit -Zymo and followed the protocol of the kit
  • Follow the instruction of the kit
 Program 

1. 98°C for 8 minutes
2. 54°C for 60 minutes
3. 4°C hold
  • Elute with 16ul elution buffer. Total volume left after elution ~15ul
  • Took 2ul for Qubit ssDNA assay
Sample Concentration in the Qubit ' uL used Dilution Sample Concentration ' Yield in 15ul (ng) % Recovery
CANC1-1 219 ng/mL 2 100 21.9 ug/mL 328.50 54.75
CANC1-2 204 ng/mL 2 100 20.4 ug/mL 306.00 51.00
CANC1-3 231 ng/mL 2 100 23.1 ug/mL 346.50 57.75
CANC1-4 209 ng/mL 2 100 20.9 ug/mL 313.50 52.25
CANC1-5 226 ng/mL 2 100 22.6 ug/mL 339.00 56.50
CANC1-6 227 ng/mL 2 100 22.7 ug/mL 340.50 56.75
CANC2-1 207 ng/mL 2 100 20.7 ug/mL 310.50 51.75
CANC2-2 195 ng/mL 2 200 19.5 ug/mL 292.50 48.75
CANC2-3 209 ng/mL 2 100 20.9 ug/mL 313.50 52.25

BSPP capture set up

  • BSC_A1 conc. = 120ng/ul
  • BSC_A2 conc. = 100ng/ul
  • GP1V4 conc. (prep 2014-05-20) = 8.37ng/ul

Probe & AmpLigase Buffer mix

Mix I. Amligase buffer and GP1V4
Components 1 rxn 18 rxn mix
GP1V4 (8.37ng/ul) 1.55 27.96
10X AmpLig Buffer 2.00 36.00
H2O 5.00 90.00
Total 8.55
Mix II. only Amligase buffer
Components 1 rxn 11 rxn mix
10X AmpLig Buffer 2.00 22.00
H2O 5.00 55.00
Total 7.00
Stoffel C1-S C2-S A1-S A2-S NTC C1 no GP1V4 A1 no GP1V4 A2 no GP1V4
Conc. (ng/ul) 21.90 20.70 120.00 100.00 20.40 120.00 100.00
Vol. for 200ng 9.13 9.66 1.67 2.00 0.00 9.80 1.67 2.00
GP1V4 (8.37ng/ul) 1.55 1.55 1.55 1.55 1.55 0.00 0.00 0.00
H2O 2.31 1.78 9.78 9.45 11.45 3.20 11.33 11.00
10X Ampligase buffer 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00
H2O in Mix 5.00 5.00 5.00 5.00 5.00 5.00 5.00 5.00
Total 20.00 20.00 20.00 20.00 20.00 20.00 20.00 20.00
Hemo Klentaq C1-S C2-S A1-S A2-S NTC C1 no GP1V4 A1 no GP1V4 A2 no GP1V4
Conc. (ng/ul) 23.10 19.50 120.00 100.00 20.90 120.00 100.00
Vol. for 200ng 8.66 10.26 1.67 2.00 0.00 9.57 1.67 2.00
GP1V4 (8.37ng/ul) 1.55 1.55 1.55 1.55 1.55 0.00 0.00 0.00
H2O 2.79 1.19 9.78 9.45 11.45 3.43 11.33 11.00
10X Ampligase buffer 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00
H2O in Mix 5.00 5.00 5.00 5.00 5.00 5.00 5.00 5.00
Total 20.00 20.00 20.00 20.00 20.00 20.00 20.00 20.00
ILMN-Pol C1-S C2-S A1-S A2-S NTC C1 no GP1V4 A1 no GP1V4 A2 no GP1V4
Conc. (ng/ul) 22.60 20.90 120.00 100.00 22.70 120.00 100.00
Vol. for 200ng 8.85 9.57 1.67 2.00 0.00 8.81 1.67 2.00
GP1V4 (8.37ng/ul) 1.55 1.55 1.55 1.55 1.55 0.00 0.00 0.00
H2O 2.60 1.88 9.78 9.45 11.45 4.19 11.33 11.00
10X Ampligase buffer 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00
H2O in Mix 5.00 5.00 5.00 5.00 5.00 5.00 5.00 5.00
Total 20.00 20.00 20.00 20.00 20.00 20.00 20.00 20.00
Program
-> 95c 30sec -> cool down to 55C at 0.02C/sec -> 55C 20h 
-> add 2ul SLN mix (2U/ul Polymerase (NEB); 0.5U/ul AmpLigase; 100uM dNTP)
-> 55C 20h-> 94C 2min -> add 2ul Exo I/III mix-> 37C 2h -> 94C 2min -> 4C hold.
  • For Illumina's polymerase I used 1ul for each sample as recommended by Xuyu

SLN mix solution