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= BSPP capture of Illumina' samples = | = BSPP capture of amplified DNA from Illumina = | ||
'''Sample list:''' | |||
* 13 samples received on 2014-03-18 [[http://genome-tech.ucsd.edu/LabNotes/index.php/Noi/NOTES/2014-3-18#BSC_samples_from_Illumina]]. Note: I processed only A1-A10 samples which are amplified DNA samples. | |||
* 8 samples received on 2014-04-14[[http://genome-tech.ucsd.edu/LabNotes/index.php/Noi/NOTES/2014-4-14#Amplified_DNA_samples_form_Illumina]] | |||
---- | |||
==== 2014-04-07 ==== | |||
== BSPP capture round1 using Polymerase from Illumina == | |||
* 2014-05-05: Received ~30ul of polymerase with property similar to Amplitaq Stoffel fragment. Conc. 3U/ul. She is not allowed to provide any further info. of this enzyme. | |||
* I performed BSPP capture on samples A1-A10 and sample #1-#8 | |||
{| {{table}} class = wikitable | |||
| align="center" style="background:#f0f0f0;"|'''Tube ID''' | |||
| align="center" style="background:#f0f0f0;"|'''Conc. (ng/ul)''' | |||
| align="center" style="background:#f0f0f0;"|'''Volume for 200/300ng (ul)''' | |||
| align="center" style="background:#f0f0f0;"|'''Tube ID''' | |||
| align="center" style="background:#f0f0f0;"|'''H2O (ul)''' | |||
| align="center" style="background:#f0f0f0;"|'''GP1V4 probe''' | |||
| align="center" style="background:#f0f0f0;"|'''10X AmpLigase Buffer''' | |||
| align="center" style="background:#f0f0f0;"|'''Total (ul)''' | |||
| align="center" style="background:#f0f0f0;"|'''Total amount (ng)''' | |||
|- | |||
| A1||189.77||1.58||A1||14.16||2.26||2.00||20.00||300.00 | |||
|- | |||
| A2||192.53||1.56||A2||14.18||2.26||2.00||20.00||300.00 | |||
|- | |||
| A3||142.63||2.10||A3||13.64||2.26||2.00||20.00||300.00 | |||
|- | |||
| A4||145.67||2.06||A4||13.68||2.26||2.00||20.00||300.00 | |||
|- | |||
| A5||182.27||1.65||A5||14.09||2.26||2.00||20.00||300.00 | |||
|- | |||
| A6||164.77||1.82||A6||13.92||2.26||2.00||20.00||300.00 | |||
|- | |||
| A7||112.93||2.66||A7||13.08||2.26||2.00||20.00||300.00 | |||
|- | |||
| A8||119.33||2.51||A8||13.23||2.26||2.00||20.00||300.00 | |||
|- | |||
| A9||188.90||1.59||A9||14.15||2.26||2.00||20.00||300.00 | |||
|- | |||
| A10||205.10||1.46||A10||14.28||2.26||2.00||20.00||300.00 | |||
|- | |||
| NTC||0.00||0.00||NTC||15.74||2.26||2.00||20.00||0.00 | |||
|- | |||
| #1||34.6||5.78||#1||9.96||2.26||2.00||20.00||200.00 | |||
|- | |||
| #2||30.4||6.58||#2||9.16||2.26||2.00||20.00||200.00 | |||
|- | |||
| #3||39.9||5.01||#3||10.73||2.26||2.00||20.00||200.00 | |||
|- | |||
| #4||41.4||4.83||#4||10.91||2.26||2.00||20.00||200.00 | |||
|- | |||
| #5||21.6||9.26||#5||6.48||2.26||2.00||20.00||200.00 | |||
|- | |||
| #6||30.9||6.47||#6||9.27||2.26||2.00||20.00||200.00 | |||
|- | |||
| #7||36.1||5.54||#7||10.20||2.26||2.00||20.00||200.00 | |||
|- | |||
| #8||34.4||5.81||#8||9.93||2.26||2.00||20.00||200.00 | |||
|- | |||
| NTC||0||0.00||NTC||15.74||2.26||2.00||20.00||0.00 | |||
|} | |||
===== GP1V4 reaction mix ===== | |||
{| {{table}} | |||
| align="center" style="background:#f0f0f0;"|'''Components''' | |||
| align="center" style="background:#f0f0f0;"|'''Volume (ul)''' | |||
| align="center" style="background:#f0f0f0;"|'''22 rxn mix''' | |||
|- | |||
| 10X AmpLigase Buffer||2.00||44.00 | |||
|- | |||
| GP1_V4 (5.75ng/ul)||2.26||49.74 | |||
|- | |||
| Toal ||'''4.26'''|| | |||
|} | |||
'''Program''' | |||
-> 95c 30sec -> cool down to 55C at 0.02C/sec -> 55C 16h | |||
-> add 3ul SLN mix (1.5U/ul '''''Illumina's polymerase''''' (3U/reaction); 0.5U/ul AmpLigase; 100uM dNTP) | |||
-> 55C 6h-> 94C 2min -> add 2ul Exo I/III mix-> 37C 2h -> 94C 2min -> 4C hold. | |||
=== SLN Mix === | |||
{| {{table}} | |||
| align="center" style="background:#f0f0f0;"|'''Components''' | |||
| align="center" style="background:#f0f0f0;"|'''Stock conc.''' | |||
| align="center" style="background:#f0f0f0;"|'''Unit''' | |||
| align="center" style="background:#f0f0f0;"|'''Final conc.''' | |||
| align="center" style="background:#f0f0f0;"|'''Unit''' | |||
| align="center" style="background:#f0f0f0;"|'''Prepare volume (ul), total 48ul''' | |||
|- | |||
| Illumina's Polymerase||3||U/ul||1.5||U/ul||24.00 | |||
|- | |||
| AmpLigase||5||U/ul||0.5||U/ul||4.80 | |||
|- | |||
| dNTP||1||mM||100||uM||4.80 | |||
|- | |||
| 10x AmpLigase Buffer||10||x||1||x||4.80 | |||
|- | |||
| H2O||||||||||9.60 | |||
|} | |||
=== Amplification === | |||
* Add 15% v/v captured DNA | |||
{| {{table}} | |||
| align="center" style="background:#f0f0f0;"|'''Components''' | |||
| align="center" style="background:#f0f0f0;"|'''1x rxn''' | |||
| align="center" style="background:#f0f0f0;"|'''22 rxn mix''' | |||
|- | |||
| Captured template||15.00||0.00 | |||
|- | |||
| 10uM AmpF6.4Sol||2.00||44.00 | |||
|- | |||
| 10uM AmpR6.3.IndX (X=1-16)||2.00||0.00 | |||
|- | |||
| 2X KAPA SYBR MM||50.00||1100.00 | |||
|- | |||
| H2O||31.00||682.00 | |||
|- | |||
| Total||100.00|| | |||
|} | |||
:- Aliquot 83, add 2ul of AmpR6.3.IndX, 15ul of template<br> | |||
:- Use the same PCR profile for library-free protocol | |||
:- Stopped reaction at 21 cycles | |||
{| {{table}} class = wikitable | |||
| align="center" style="background:#f0f0f0;"|'''Samples''' | |||
| align="center" style="background:#f0f0f0;"|'''Index''' | |||
| align="center" style="background:#f0f0f0;"|'''Samples''' | |||
| align="center" style="background:#f0f0f0;"|'''Index''' | |||
| align="center" style="background:#f0f0f0;"|'''Samples''' | |||
| align="center" style="background:#f0f0f0;"|'''Index''' | |||
|- | |||
| A1||Ind_41||A9||Ind_49||#1||Ind_57 | |||
|- | |||
| A2||Ind_42||A10||Ind_50||#2||Ind_58 | |||
|- | |||
| A3||Ind_43||NTC-1||Ind_51||#3||Ind_59 | |||
|- | |||
| A4||Ind_44||NTC-2||Ind_52||#4||Ind_60 | |||
|- | |||
| A5||Ind_45||NTC-PCR||Ind_53||#5||Ind_61 | |||
|- | |||
| A6||Ind_46||||||#6||Ind_62 | |||
|- | |||
| A7||Ind_47||||||#7||Ind_63 | |||
|- | |||
| A8||Ind_48||||||#8||Ind_64 | |||
|} | |||
* Verified PCR product in 6% TBE gel by loading 4ul of PCR product | |||
[[File:ZhangLab_2 2014-05-09 15hr 25min_ILMN_GP1V4_gel1_A1-A10_NTC.jpg| 400px]] [[File:ZhangLab_2 2014-05-09 15hr 27min_ILMN_GP1V4_gel2#1-8_NTC.jpg| 400px]] | |||
* Perform AMPure bead purification (0.7X volume) | |||
* Elute with 55ul EB buffer | |||
* Verify and quantify DNA concentration in 6% TBE gel by loading 4ul of bead-purified DNA | |||
[[File:ZhangLab_2 2014-05-17 17hr 52min_PQ4ul_A1-A10_Exp5-07.jpg| 400px]] [[File:ZhangLab_2 2014-05-17 17hr 52min_PQ4ul_#1-#8_NTC_Exp5-07.jpg|400px]] |
Revision as of 18:50, 18 May 2014
BSPP capture of amplified DNA from Illumina
Sample list:
- 13 samples received on 2014-03-18 [[1]]. Note: I processed only A1-A10 samples which are amplified DNA samples.
- 8 samples received on 2014-04-14[[2]]
2014-04-07
BSPP capture round1 using Polymerase from Illumina
- 2014-05-05: Received ~30ul of polymerase with property similar to Amplitaq Stoffel fragment. Conc. 3U/ul. She is not allowed to provide any further info. of this enzyme.
- I performed BSPP capture on samples A1-A10 and sample #1-#8
Tube ID | Conc. (ng/ul) | Volume for 200/300ng (ul) | Tube ID | H2O (ul) | GP1V4 probe | 10X AmpLigase Buffer | Total (ul) | Total amount (ng) |
A1 | 189.77 | 1.58 | A1 | 14.16 | 2.26 | 2.00 | 20.00 | 300.00 |
A2 | 192.53 | 1.56 | A2 | 14.18 | 2.26 | 2.00 | 20.00 | 300.00 |
A3 | 142.63 | 2.10 | A3 | 13.64 | 2.26 | 2.00 | 20.00 | 300.00 |
A4 | 145.67 | 2.06 | A4 | 13.68 | 2.26 | 2.00 | 20.00 | 300.00 |
A5 | 182.27 | 1.65 | A5 | 14.09 | 2.26 | 2.00 | 20.00 | 300.00 |
A6 | 164.77 | 1.82 | A6 | 13.92 | 2.26 | 2.00 | 20.00 | 300.00 |
A7 | 112.93 | 2.66 | A7 | 13.08 | 2.26 | 2.00 | 20.00 | 300.00 |
A8 | 119.33 | 2.51 | A8 | 13.23 | 2.26 | 2.00 | 20.00 | 300.00 |
A9 | 188.90 | 1.59 | A9 | 14.15 | 2.26 | 2.00 | 20.00 | 300.00 |
A10 | 205.10 | 1.46 | A10 | 14.28 | 2.26 | 2.00 | 20.00 | 300.00 |
NTC | 0.00 | 0.00 | NTC | 15.74 | 2.26 | 2.00 | 20.00 | 0.00 |
#1 | 34.6 | 5.78 | #1 | 9.96 | 2.26 | 2.00 | 20.00 | 200.00 |
#2 | 30.4 | 6.58 | #2 | 9.16 | 2.26 | 2.00 | 20.00 | 200.00 |
#3 | 39.9 | 5.01 | #3 | 10.73 | 2.26 | 2.00 | 20.00 | 200.00 |
#4 | 41.4 | 4.83 | #4 | 10.91 | 2.26 | 2.00 | 20.00 | 200.00 |
#5 | 21.6 | 9.26 | #5 | 6.48 | 2.26 | 2.00 | 20.00 | 200.00 |
#6 | 30.9 | 6.47 | #6 | 9.27 | 2.26 | 2.00 | 20.00 | 200.00 |
#7 | 36.1 | 5.54 | #7 | 10.20 | 2.26 | 2.00 | 20.00 | 200.00 |
#8 | 34.4 | 5.81 | #8 | 9.93 | 2.26 | 2.00 | 20.00 | 200.00 |
NTC | 0 | 0.00 | NTC | 15.74 | 2.26 | 2.00 | 20.00 | 0.00 |
GP1V4 reaction mix
Components | Volume (ul) | 22 rxn mix |
10X AmpLigase Buffer | 2.00 | 44.00 |
GP1_V4 (5.75ng/ul) | 2.26 | 49.74 |
Toal | 4.26 |
Program -> 95c 30sec -> cool down to 55C at 0.02C/sec -> 55C 16h -> add 3ul SLN mix (1.5U/ul Illumina's polymerase (3U/reaction); 0.5U/ul AmpLigase; 100uM dNTP) -> 55C 6h-> 94C 2min -> add 2ul Exo I/III mix-> 37C 2h -> 94C 2min -> 4C hold.
SLN Mix
Components | Stock conc. | Unit | Final conc. | Unit | Prepare volume (ul), total 48ul |
Illumina's Polymerase | 3 | U/ul | 1.5 | U/ul | 24.00 |
AmpLigase | 5 | U/ul | 0.5 | U/ul | 4.80 |
dNTP | 1 | mM | 100 | uM | 4.80 |
10x AmpLigase Buffer | 10 | x | 1 | x | 4.80 |
H2O | 9.60 |
Amplification
- Add 15% v/v captured DNA
Components | 1x rxn | 22 rxn mix |
Captured template | 15.00 | 0.00 |
10uM AmpF6.4Sol | 2.00 | 44.00 |
10uM AmpR6.3.IndX (X=1-16) | 2.00 | 0.00 |
2X KAPA SYBR MM | 50.00 | 1100.00 |
H2O | 31.00 | 682.00 |
Total | 100.00 |
- - Aliquot 83, add 2ul of AmpR6.3.IndX, 15ul of template
- - Use the same PCR profile for library-free protocol
- - Stopped reaction at 21 cycles
Samples | Index | Samples | Index | Samples | Index |
A1 | Ind_41 | A9 | Ind_49 | #1 | Ind_57 |
A2 | Ind_42 | A10 | Ind_50 | #2 | Ind_58 |
A3 | Ind_43 | NTC-1 | Ind_51 | #3 | Ind_59 |
A4 | Ind_44 | NTC-2 | Ind_52 | #4 | Ind_60 |
A5 | Ind_45 | NTC-PCR | Ind_53 | #5 | Ind_61 |
A6 | Ind_46 | #6 | Ind_62 | ||
A7 | Ind_47 | #7 | Ind_63 | ||
A8 | Ind_48 | #8 | Ind_64 |
- Verified PCR product in 6% TBE gel by loading 4ul of PCR product
File:ZhangLab 2 2014-05-09 15hr 25min ILMN GP1V4 gel1 A1-A10 NTC.jpg File:ZhangLab 2 2014-05-09 15hr 27min ILMN GP1V4 gel2
- Perform AMPure bead purification (0.7X volume)
- Elute with 55ul EB buffer
- Verify and quantify DNA concentration in 6% TBE gel by loading 4ul of bead-purified DNA
File:ZhangLab 2 2014-05-17 17hr 52min PQ4ul A1-A10 Exp5-07.jpg File:ZhangLab 2 2014-05-17 17hr 52min PQ4ul