Noi/NOTES/2014-6-10: Difference between revisions
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| PC-T-6||Ind_54||1.13||39.70||26.45 | | PC-T-6||Ind_54||1.13||39.70||26.45 | ||
|- | |- | ||
| PC- | | PC-T-7||Ind_55||2.99||104.62||10.04 | ||
|} | |} | ||
* Purified in 2 well of 5-well 6% TBE PAGE | * Purified in 2 well of 5-well 6% TBE PAGE | ||
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* '''Library ID: NP-BSPP-V1N3-TSP-Jun10''' --> was changed to '''NP-BSPP-V1N3-TSP-Jun13'''<br> | * '''Library ID: NP-BSPP-V1N3-TSP-Jun10''' --> was changed to '''NP-BSPP-V1N3-TSP-Jun13'''<br> | ||
* Conc. by Qubit dsDNA HS quantification: 0.58ng/ul | * Conc. by Qubit dsDNA HS quantification: 0.58ng/ul | ||
* Handed sequencing libraries to Alan | * Handed sequencing libraries to Alan | ||
==== Index for serum samples ==== | ==== Index for serum samples ==== | ||
* I used barcoded primers in column #6 | * I used barcoded primers in column #6 |
Latest revision as of 22:02, 19 September 2014
MONOD V1N3 BSPP capture of serum and solid tumor DNA[edit]
serum DNA[edit]
- Some samples had volume >20ul, I evaporated to reduce the volume (~from 30ul --> 13/14ul, 30C for 10min)
Sample | Original conc. (ng/ul) | Original vol. before evap. | Volume for 50ng (ul) | H2O (ul) | Lightning Conv. Reagent (ul) | Total volume (ul) | Total DNA amount (ng) |
PC-S_2 (evap) | 1.66 | 27.00 | 14.00 | 6.00 | 130.00 | 150.00 | 44.82 |
PC-S_3 | 21.40 | NA | 2.34 | 17.66 | 130.00 | 150.00 | 50.00 |
PC-S_5 (evap) | 1.58 | 27.00 | 14.00 | 6.00 | 130.00 | 150.00 | 42.66 |
PC-S_6 | 17.50 | NA | 2.86 | 17.14 | 130.00 | 150.00 | 50.00 |
PC-S_7 (evap) | 1.80 | 25.00 | 14.00 | 6.00 | 130.00 | 150.00 | 45.00 |
PC-S_9 | 5.72 | NA | 8.74 | 11.26 | 130.00 | 150.00 | 50.00 |
PC-S_10 (evap) | 1.71 | 27.00 | 13.00 | 7.00 | 130.00 | 150.00 | 46.17 |
Solid tumor tissue DNA[edit]
Sample | Conc. (ng/ul) | Volume for 50ng (ul) | H2O (ul) | Lightning Conv. Reagent (ul) | Total volume (ul) | Total DNA amount (ng) |
PC-T-1 | 82.70 | 0.60 | 19.40 | 130.00 | 150.00 | 50.00 |
PC-T-2 | 35.80 | 1.40 | 18.60 | 130.00 | 150.00 | 50.00 |
PC-T-4 | 42.80 | 1.17 | 18.83 | 130.00 | 150.00 | 50.00 |
PC-T-6 | 15.20 | 3.29 | 16.71 | 130.00 | 150.00 | 50.00 |
PC-C-7 | 16.30 | 3.07 | 16.93 | 130.00 | 150.00 | 50.00 |
- I did the 2nd set for those samples with high amount of DNA. Just in case there is something wrong with the result of some samples.
Sample | Conc. (ng/ul) | Volume for 50ng (ul) | H2O (ul) | Lightning Conv. Reagent (ul) | Total volume (ul) | Total DNA amount (ng) |
PC-S_3 | 21.40 | 2.34 | 17.66 | 130.00 | 150.00 | 50.00 |
PC-S_6 | 17.50 | 2.86 | 17.14 | 130.00 | 150.00 | 50.00 |
PC-S_9 | 5.72 | 8.74 | 11.26 | 130.00 | 150.00 | 50.00 |
PC-T-1 | 82.70 | 0.60 | 19.40 | 130.00 | 150.00 | 50.00 |
PC-T-2 | 35.80 | 1.40 | 18.60 | 130.00 | 150.00 | 50.00 |
PC-T-4 | 42.80 | 1.17 | 18.83 | 130.00 | 150.00 | 50.00 |
PC-T-6 | 15.20 | 3.29 | 16.71 | 130.00 | 150.00 | 50.00 |
PC-C-7 | 16.30 | 3.07 | 16.93 | 130.00 | 150.00 | 50.00 |
- Elute BIS-DNA with 9.2ul Elution buffer and expected DNA volume left ~8.4ul for capture, but I only have DNA left 7ul. Next time, I will elute with ~10ul Elution buffer.
BSPP capture set up[edit]
- Since the input DNA for BIS-CVT was quite low, I did not measure BIS-DNA concentration since I did not want to lose DNA. I assumed that there was DNA after BIS-CVT ~10ng from 20% recovery rate.
- I will use probe to target ration 1000:1 --> requires V1N3 amount 0.44ug from 10X diluted V1N3 (0.73ng/ul)
strip 1 | PC-S_2 | PC-S_3 | PC-S_5 | PC-S_6 | PC-S_7 | PC-S_9 | PC-S_10 |
Volume (ul) | 7.00 | 7.00 | 7.00 | 7.00 | 7.00 | 7.00 | 7.00 |
V1N3 amount (ng) | 0.44 | 0.44 | 0.44 | 0.44 | 0.44 | 0.44 | 0.44 |
V1N3 conc. (ng/ul) | 0.73 | 0.73 | 0.73 | 0.73 | 0.73 | 0.73 | 0.73 |
V1N3 volume (ul) | 0.60 | 0.60 | 0.60 | 0.60 | 0.60 | 0.60 | 0.60 |
H2O in buffer mix | 1.40 | 1.40 | 1.40 | 1.40 | 1.40 | 1.40 | 1.40 |
10X Ampligase Buffer | 1.00 | 1.00 | 1.00 | 1.00 | 1.00 | 1.00 | 1.00 |
Total | 10.00 | 10.00 | 10.00 | 10.00 | 10.00 | 10.00 | 10.00 |
strip 2 | PC-T-1 | PC-T-2 | PC-T-4 | PC-T-6 | PC-C-7 | NTC |
Volume (ul) | 7.00 | 7.00 | 7.00 | 7.00 | 7.00 | 7.00 |
V1N3 amount (ng) | 0.44 | 0.44 | 0.44 | 0.44 | 0.44 | 0.44 |
V1N3 conc. (ng/ul) | 0.73 | 0.73 | 0.73 | 0.73 | 0.73 | 0.73 |
V1N3 volume (ul) | 0.60 | 0.60 | 0.60 | 0.60 | 0.60 | 0.60 |
10X Ampligase Buffer | 1.00 | 1.00 | 1.00 | 1.00 | 1.00 | 1.00 |
H2O in buffer mix | 1.40 | 1.40 | 1.40 | 1.40 | 1.40 | 1.40 |
Total | 10.00 | 10.00 | 10.00 | 10.00 | 10.00 | 10.00 |
extra set[edit]
- With the same condition as samples above
PC-S_3 | PC-S_6 | PC-S_9 | PC-T-1 | PC-T-2 | PC-T-4 | PC-T-6 | PC-C-7 |
V1N3 & 10X Ampligase Buffer Mix[edit]
Components | 1x rxn | 25x rxn |
V1N3 (0.73ng/ul) | 0.60 | 15.00 |
10X Ampligase Buffer Mix | 1.00 | 25.00 |
H2O | 1.40 | 35.00 |
Total | 3.00 |
Program
-> 95c 30sec -> cool down to 55C at 0.02C/sec -> 55C 20h -> add 1ul KLN mix (2U/ul Hemo Klentaq; 0.5U/ul AmpLigase; 100uM dNTP) -> 55C 6h-> 94C 2min -> add 1.5ul Exo I/III mix (1ul of 20U/ul ExoI & 0.5ul of 200U/ul of ExoIII)-> 37C 2h -> 94C 5min -> 4C hold.
KLN Solution Mix[edit]
Components | Stock conc. | Unit | Final conc. | Unit | Prepare volume total 30ul |
Hemo KlenTaq | 100 | % | 20 | % | 6.00 |
AmpLigase | 5 | U/ul | 0.5 | U/ul | 3.00 |
dNTP | 1 | mM | 100 | uM | 3.00 |
10x AmpLigase Buffer | 10 | x | 1 | x | 3.00 |
H2O | 15.00 |
Amplification[edit]
- Used the same condition for PCR as experiment on 2014-06-02 with 10ul captured DNA in total 75ul
Components | 1x rxn | 22.5 rxn mix |
Captured template | 10.00 | 0.00 |
10uM AmpF6.4Sol | 1.50 | 33.75 |
10uM AmpR6.3.IndX | 1.50 | 0.00 |
2X KAPA SYBR MM | 37.50 | 843.75 |
H2O | 24.50 | 551.25 |
Total | 75.00 |
Program (Eppendorf Realplex) 95C 30s -> (95C 10s -> 58C 20s -> 72C 20s)x8 -> (95C 10s -> 72C 20s)x19 -> 72C 3min
quantification of individual sequencing libraries[edit]
V1N3-S File:ZhangLab 2 2014-06-12 15hr 10min PC-S V1N3.jpg I did not include index43 in the library pools as the concentration is too low.
Sample | Index | Conc. (ng/ul) | Yield in 35ul (ng) | Volume for 20ng (ul) |
PC-S_2 | Ind_41 | 2.26 | 79.04 | 8.86 |
PC-S_3 | Ind_42 | 1.11 | 38.78 | 18.05 |
PC-S_6 | Ind_44 | 0.71 | 24.86 | 28.16 |
PC-S_7 | Ind_45 | 1.39 | 48.75 | 14.36 |
PC-S_9 | Ind_46 | 0.75 | 26.14 | 26.78 |
PC-S_10 | Ind_47 | 0.78 | 27.47 | 25.48 |
- Purified in 2 well of 5-well 6% TBE PAGE
V1N3-T File:ZhangLab 2 2014-06-12 15hr 10min PC-T V1N3.jpg
Sample | Index | Conc. (ng/ul) | Yield in 35ul (ng) | Volume for 30ng (ul) |
PC-T-1 | Ind_51 | 2.99 | 104.81 | 10.02 |
PC-T-2 | Ind_52 | 2.50 | 87.33 | 12.02 |
PC-T-4 | Ind_53 | 2.96 | 103.45 | 10.15 |
PC-T-6 | Ind_54 | 1.13 | 39.70 | 26.45 |
PC-T-7 | Ind_55 | 2.99 | 104.62 | 10.04 |
- Purified in 2 well of 5-well 6% TBE PAGE
NP-BSPP-V1N3-S-Jun10, 2014 NP-BSPP-V1N3-T-Jun10, 2014 File:ZhangLab 2 2014-06-13 10hr 19min V1N3 serum PAGE-SS.jpg File:ZhangLab 2 2014-06-13 10hr 19min V1N3 tumor PAGE-SS.jpg
- Since we are going to get sequencing at Illumina, I pooled three library pools to one tube with the same molar ratio
Library pool # of libraries Ratio toV1N3-P Conc.by Qubit dsDNA HS (ng/ul) Pool 4ng of V1N3-P (ul) V1N3-S 6 1.500 0.590 10.169 V1N3-T 5 1.250 0.610 8.197 V1N3-P 4 1.000 0.488 8.197
- Library ID: NP-BSPP-V1N3-TSP-Jun10 --> was changed to NP-BSPP-V1N3-TSP-Jun13
- Conc. by Qubit dsDNA HS quantification: 0.58ng/ul
- Handed sequencing libraries to Alan
Index for serum samples[edit]
- I used barcoded primers in column #6
strip 1 | PC-S_2 | PC-S_3 | PC-S_5 | PC-S_6 | PC-S_7 | PC-S_9 | PC-S_10 |
Index | Ind_41 | Ind_42 | Ind_43 | Ind_44 | Ind_45 | Ind_46 | Ind_47 |
- I used barcoded primers in column #7 and start at well C1 (= Ind_51) to avoid bad barcodes
Project ID | Sample type | Zhang lab ID | Index |
UCSD-004-05 | Serum | PC-S_2 | Ind_41 |
UCSD-004-06 | Serum | PC-S_3 | Ind_42 |
UCSD-004-08 | Serum | PC-S_5 | Ind_43 |
UCSD-004-09 | Serum | PC-S_6 | Ind_44 |
UCSD-004-10 | Serum | PC-S_7 | Ind_45 |
UCSD-004-12 | Serum | PC-S_9 | Ind_46 |
UCSD-004-13 | Serum | PC-S_10 | Ind_47 |
Index for solid tumor tissue samples[edit]
strip 2 | PC-T-1 | PC-T-2 | PC-T-4 | PC-T-6 | PC-T-7 | NTC |
Index | Ind_51 | Ind_52 | Ind_53 | Ind_54 | Ind_55 | Ind_56 |
Project ID | Sample type | Zhang lab ID | Index |
UCSD-004-04 | Solid tumor tissue | PC-T-1 | Ind_51 |
UCSD-004-05 | Solid tumor tissue | PC-T-2 | Ind_52 |
UCSD-004-07 | Solid tumor tissue | PC-T-4 | Ind_53 |
UCSD-004-09 | Solid tumor tissue | PC-T-6 | Ind_54 |
UCSD-004-10 | Solid tumor tissue | PC-C-7 | Ind_55 |
Index for plasma sample from Kang Zhang's lab with pre-amplification[edit]
- Lab note on 2014-06-07: [[1]]
Index | Sample | Conc. (ng/ul) | Yield in 70ul (ng) | Volume for 40ng (ul) |
Ind_33 | NC-P-5ng_1 | 1.00 | 69.68 | 40.19 |
Ind_34 | NC-P-5ng_2 | 0.62 | 43.70 | 50.00** |
Ind_35 | NC-P-10ng_1 | 0.88 | 61.38 | 45.62 |
Ind_36 | NC-P-10ng_2 | 1.26 | 88.54 | 31.63 |
- MiSeq data: /home/kunzhang/seqStore/20140613_MiSeq