Rui:LabNotes/Haplotyping/2014-12-2: Difference between revisions
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LMS_selector V2 (1ng/ul: 33nM) 20.00 0.4ng/ul ratio 200:1 | LMS_selector V2 (1ng/ul: 33nM) 20.00 0.4ng/ul ratio 200:1 | ||
linkerV2 or linkerV2_UMI (100nM) 20.00 3:1 linker:selector | linkerV2 or linkerV2_UMI (100nM) 20.00 3:1 linker:selector | ||
10X AmpLigase buffer 5.00 1x | 10X AmpLigase buffer 5.00 1x | ||
H2O 5.00 | H2O 5.00 | ||
Total 50.00 | Total 50.00 |
Revision as of 09:04, 4 December 2014
Purpose
- repeat selector procedure:
- selector : gDNA = 200:1; linker : selector = 3:1
- direct gDNA input: 12, 24, 48ng
- compare selector vs padlock probe
- premade
Samples
- HapMap gDNA from Coriell GM18506 (diluted to 50ng/ul)
gDNA input | Selector set | Probe ratio | Indx | Test | |
#1 | 48ng | linkerV2_UMI | 200:1; 3:1 | Indx01 | selector_UMI |
#2 | 24ng | linkerV2_UMI | 200:1; 3:1 | Indx02 | selector_UMI |
#3 | 12ng | linkerV2_UMI | 200:1; 3:1 | Indx03 | selector_UMI |
#4 | NTC | linkerV2_UMI | 200:1; 3:1 | Indx04 | selector_UMI |
#5 | 48ng | linkerV2 | 200:1; 3:1 | Indx05 | selector |
#6 | 24ng | linkerV2 | 200:1; 3:1 | Indx06 | selector |
#7 | 12ng | linkerV2 | 200:1; 3:1 | Indx07 | selector |
#8 | NTC | linkerV2 | 200:1; 3:1 | Indx08 | selector |
#9 | 48ng | linkerV2 | 200:1 | Indx09 | padlock probe |
#10 | 24ng | linkerV2 | 200:1 | Indx10 | padlock probe |
#11 | 12ng | linkerV2 | 200:1 | Indx11 | padlock probe |
#12 | NTC | linkerV2 | 200:1 | Indx12 | padlock probe |
#13 | 50ng_digested (Kun's, asPC) | linkerV2_UMI | 200:1; 3:1 | Indx13 | selector_UMI |
#14 | 20ng_digested (Kun's, asPC) | linkerV2_UMI | 200:1; 3:1 | Indx14 | selector_UMI |
Procedure
1a. R.E digestion
Components | 48ng | 24ng | 12ng | NTC |
gDNA (12ng/ul) | 4.00 | 2.00 | 1.00 | 0.00 |
10x cutsmart buffer | 1.00 | 1.00 | 1.00 | 1.00 |
DpnII (50U/ul) -> dilute to 5U/ul | 2.50 | 1.25 | 0.63 | 0.63 |
BfaI (10U/ul) -> dilute to 2U/ul | 2.50 | 1.25 | 0.63 | 0.63 |
H2O | 0.00 | 4.50 | 6.75 | 7.75 |
Total | 10.00 | 10.00 | 10.00 | 10.00 |
Temperature | Duration |
Digestion | 37C, 1hr |
hold | 4C |
Components | Volume (ul) | Final conc. |
10X NEBuffer 3.1 | 1 | 1x |
Temperature | Duration |
Digestion | 37C, 30min |
heat inactivation | 80C, 20min |
hold | 4C |
1b. Assembly of selecotr and linker
Components Vol. (ul) Final LMS_selector V2 (1ng/ul: 33nM) 20.00 0.4ng/ul ratio 200:1 linkerV2 or linkerV2_UMI (100nM) 20.00 3:1 linker:selector 10X AmpLigase buffer 5.00 1x H2O 5.00 Total 50.00
- 94C, 2min -> 37C, 10min
2. Hybridization: Ampligase buffer needs to be aliquot and stored individually
Components | Final conc. | 48ng | 24ng | 12ng | NTC | 48ng | 24ng | 12ng | NTC |
digested gDNA | 0/12/24/48ng | 10.00 | 10.00 | 10.00 | 10.00 | 4.00 | 2.00 | 1.00 | 0.00 |
10x Ampligase buffer | 1x | 1.36 | 1.68 | 1.84 | 1.84 | 2.1 | 2.1 | 2.1 | 2.1 |
MgCl2 (25mM) | 2mM | 1.60 | 1.60 | 1.60 | 1.60 | 0.00 | 0.00 | 0.00 | 0.00 |
NAD (50mM) | 1mM | 0.40 | 0.40 | 0.40 | 0.40 | 0.00 | 0.00 | 0.00 | 0.00 |
Selector/linker | 200:1 | 6.38 | 3.20 | 1.60 | 1.60 | 2.55 | 1.28 | 0.64 | 0.64 |
H2O | 0.26 | 3.12 | 4.56 | 4.56 | 11.35 | 14.62 | 16.26 | 17.26 | |
Total | 20.00 | 20.00 | 20.00 | 20.00 | 20.00 | 20.00 | 20.00 | 20.00 |
Thermocycler steps | Duration |
Denature | 95C, 5min |
Cool down to 60C slowely | at -0.2C/sec |
Hold | 60C, 16hrs |
3. Synthesis: Ampligase plus Taq pol.
Components | Selector | Probe |
Ampligase (2.5U/ul diluted in water) | 1 | 0.4 |
HemoKlen Taq (10U/ul) | 0 | 0.4 |
dNTP mix (1mM) | 0 | 0.2 |
Total | 1 | 1 |
Thermocycler steps | Duration |
Synthesis | 60C, 2hrs |
Denature | 94C, 2min |
hold | 8C |
4. ExoM: made mixture of ExoI/III before adding into samples
Components | Volume (ul) | Final conc. |
ExoI | 1 | |
ExoIII | 1 | |
Total | 2 |
Thermocycler steps | Duration |
Digestion | 37C, 1hr |
Denature | 80C, 20min |
hold | 8C |
5. PCR amplification: quick run with 1/10 of sample for an estimation of capture efficiency
- Sample PCR with Indexes
Components | Volumn (ul) | Final conc. |
cirDNA template | 22 | all |
Q5 MM | 30 | 1x |
10 uM CP-2-RA | 1.2 | 0.2uM |
10 uM AmpR6.3.Indx | 1.2 | 0.2uM |
20x SYBR | 0.5 | 0.2x |
H2O | 5.09999999999999 | |
Total | 60 |
Realtime-PCR profile | QPCR cycles |
98C 5min | 1 |
98C 15sec -> 58C 30sec -> 72C 15sec | 8 |
98C 15sec -> 72C 15sec | 16 |
72C 3min | 1 |
Hold 10C |
6. Beads purfication on PCR amplicons
Components | Volume (ul) | Final conc. |
PCR amplicon | 55 | 5ul for gel |
AMPure beads | 50 | 0.8X |
* Clearly remove majority of bands/smear below 200bp |
Results
- QPCR curve
File:Nv4 1-4 QPCR.jpgFile:Nv4 5-12 QPCR.jpg
- PAGE check
File:Nv4 1-4 gel.jpgFile:Nv4 5-12 gel.jpg
Mixed lib.
- Following libraries were combined and size selected for sequencing
- Tube label: RL-Nv4_UMI-Dec3
gDNA input | Selector set | Probe ratio | Indx | Test | Mixed V (ul) | |
#1 | 48ng | linkerV2_UMI | 200:1; 3:1 | Indx01 | selector_UMI | 2 |
#2 | 24ng | linkerV2_UMI | 200:1; 3:1 | Indx02 | selector_UMI | 4 |
#3 | 12ng | linkerV2_UMI | 200:1; 3:1 | Indx03 | selector_UMI | 8 |
#13 | 50ng_digested (Kun's, asPC) | linkerV2_UMI | 200:1; 3:1 | Indx13 | selector_UMI | 2 |
#14 | 20ng_digested (Kun's, asPC) | linkerV2_UMI | 200:1; 3:1 | Indx14 | selector_UMI | 4 |
* size selection can be skipped, pretty clean below 200bp |
- Tube label: RL-Nv4-Dec3
gDNA input | Selector set | Probe ratio | Indx | Test | Mixed V (ul) | |
#5 | 48ng | linkerV2 | 200:1; 3:1 | Indx05 | selector | 2 |
#6 | 24ng | linkerV2 | 200:1; 3:1 | Indx06 | selector | 4 |
#7 | 12ng | linkerV2 | 200:1; 3:1 | Indx07 | selector | 8 |
#9 | 48ng | linkerV2 | 200:1 | Indx09 | padlock probe | 2 |
#10 | 24ng | linkerV2 | 200:1 | Indx10 | padlock probe | 4 |
#11 | 12ng | linkerV2 | 200:1 | Indx11 | padlock probe | 8 |
* need size selection, still have a few bands below 200bp |