Dinh/NOTES/2010-7-3: Difference between revisions
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>Dinh m (Created page with '==Sanger sequencing of PCR product with probe linker== ===Primers=== LinkerR-LMNA-F: GTT GGA GGC TCA TCG TTC CTA TTT TGG GCC TGA GTG GTC AGT C LinkerL-LMNA-R: GAC CCG CCT GCA …') |
>Dinh mNo edit summary |
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94C 2min | 94C 2min | ||
94C 30s | 94C 30s | ||
62C 40s | |||
72C 40s | 72C 40s | ||
Go to 2 for 7x | Go to 2 for 7x |
Revision as of 20:58, 2 July 2010
Sanger sequencing of PCR product with probe linker
Primers
LinkerR-LMNA-F: GTT GGA GGC TCA TCG TTC CTA TTT TGG GCC TGA GTG GTC AGT C LinkerL-LMNA-R: GAC CCG CCT GCA GGA TTT GGC AGG CAG ATG TTA TCG AGG TCC GAC Product length: 373+32bp = 405bp Position: chr1:156,108,195-156,108,590
PCR
Template (1-2ng/ul): 3ul F primer (10uM) : 1ul R primer (10uM) : 1ul H20 : 20ul Taq 2x MM : 25ul ----------------------- 50ul Templates used: BJ-ips, HGPS-ips, HGPS72
Program: 94C 2min 94C 30s 62C 40s 72C 40s Go to 2 for 7x 94C 30s 72C 40s Go to 7 for 23x 72C 3min 15C forever
Purify by 0.9x volumes of Agencourt beads.
- Quantification by Nanodrop
- Premix for Genewiz sequencing: