Rui:LabNotes/Haplotyping/2014-12-2: Difference between revisions
Jump to navigation
Jump to search
>RuiLiu |
>RuiLiu |
||
Line 233: | Line 233: | ||
===1b. Assembly of selecotr and linker === | ===1b. Assembly of selecotr and linker === | ||
{| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8<\hiddentext> | |||
|- style="background-color:#D9D9D9;font-size:10pt" valign="bottom" | |||
| width="192" height="15" | Pre-made selector/linker | |||
| width="72" | Vol. (ul) | |||
| width="72" | Final | |||
| align="center" width="79" | | |||
===2. Hybridization: Ampligase buffer needs to be aliquot and stored individually=== | ===2. Hybridization: Ampligase buffer needs to be aliquot and stored individually=== |
Revision as of 08:46, 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
Pre-made selector/linker | Vol. (ul) | Final |
2. Hybridization: Ampligase buffer needs to be aliquot and stored individually
3. Synthesis: Ampligase plus Taq pol.
4. ExoM: made mixture of ExoI/III before adding into samples
5. PCR amplification: quick run with 1/10 of sample for an estimation of capture efficiency
6. Beads purfication on PCR amplicons
Results
Mixed lib.
|