Rui:LabNotes/Haplotyping/2011-8-17: Difference between revisions
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>RuiLiu (Created page with 'File:8.17.11_regularHap.jpg File:8.17.11_1ul.jpg File:8.17.11_0.4ul.jpg File:8.17.11_0.2ul.jpg') |
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==Testing for haplotyping method== | |||
* First test: protease treatment after tagmentation | |||
I once tested it as Athurva used (1ul, 1:100 dilution) , however it showed delayed amplification. | |||
This time, I reduced the protease (0.4ul and 0.2ul vs 1ul), as I used less transposon (0.4, 1:50d) than others (1ul, 1:50d). | |||
* Second test: 2nd round of PCR with beads or completely purified DNA | |||
===Procedure=== | |||
* cells aliquot on 8/8/11 [http://genome-tech.ucsd.edu/LabNotes/index.php/Rui:LabNotes/Haplotyping/2011-8-8] | |||
* 1 ul of 10 cells/ul + 30ul lysis buffer (1X, made on 8/3/11 [http://genome-tech.ucsd.edu/LabNotes/index.php/Rui:LabNotes/Haplotyping/2011-8-3]); 37C, 30min | |||
* Add 0.3ul protease to tube, immediately aliquot 1ul/tube for 20 tubes; 50C, 15min, 70C, 15min | |||
* Tagmentation with 0.4ul of 1:50d transposon | |||
* Protease treatment: none, 0.2ul (1:100d), 0.4ul (1:100d), 1ul (1:100d); 50C, 10min, 70C, 20min | |||
* PCR with blue/orange (mixed primers tube and new Kapa master mix) | |||
===QPCR curve=== | |||
None treatment | |||
[[File:8.17.11_regularHap.jpg]] | [[File:8.17.11_regularHap.jpg]] | ||
+0.2ul protease | |||
[[File:8.17.11_0.2ul.jpg]] | |||
+0.4ul protease | |||
[[File:8.17.11_0.4ul.jpg]] | |||
+1ul protease | |||
[[File:8.17.11_1ul.jpg]] | [[File:8.17.11_1ul.jpg]] | ||
[[File:8. | ===TBE PAGE check=== | ||
[[File:8.18.11_0-0.2.jpg]] | |||
[[File:8.18.11_0.4-1.jpg]] |
Revision as of 19:43, 18 August 2011
Testing for haplotyping method
- First test: protease treatment after tagmentation
I once tested it as Athurva used (1ul, 1:100 dilution) , however it showed delayed amplification. This time, I reduced the protease (0.4ul and 0.2ul vs 1ul), as I used less transposon (0.4, 1:50d) than others (1ul, 1:50d).
- Second test: 2nd round of PCR with beads or completely purified DNA
Procedure
- cells aliquot on 8/8/11 [1]
- 1 ul of 10 cells/ul + 30ul lysis buffer (1X, made on 8/3/11 [2]); 37C, 30min
- Add 0.3ul protease to tube, immediately aliquot 1ul/tube for 20 tubes; 50C, 15min, 70C, 15min
- Tagmentation with 0.4ul of 1:50d transposon
- Protease treatment: none, 0.2ul (1:100d), 0.4ul (1:100d), 1ul (1:100d); 50C, 10min, 70C, 20min
- PCR with blue/orange (mixed primers tube and new Kapa master mix)
QPCR curve
None treatment File:8.17.11 regularHap.jpg
+0.2ul protease File:8.17.11 0.2ul.jpg
+0.4ul protease File:8.17.11 0.4ul.jpg
+1ul protease File:8.17.11 1ul.jpg