Sean Lauber:Kras G12D Genotyping: Difference between revisions
From OpenWetWare
Jump to navigationJump to search
(New page: This protocol makes use of the DirectPCR mouse tail lysis reagent (Viagen, cat 102-T) to obtain template and the hot start Platinum Taq (Invitrogen, cat 10966-026). 1 mm tail ends are co...) |
No edit summary |
||
| Line 1: | Line 1: | ||
This protocol makes use of the DirectPCR mouse tail lysis reagent (Viagen, cat 102-T) to obtain template and the | This protocol makes use of the DirectPCR mouse tail lysis reagent (Viagen, cat 102-T) to obtain template and the hot start Platinum Taq (Invitrogen, cat 10966-026). 1 mm tail ends are collected from mice (typically at 4-6 weeks) and the tail is degraded using the DirectPCR reagent before use in PCR. | ||
1. 125 ul of DirectPCR reagent and 4.73 ul of Proteinase K are added to each tube containing a 1 mm tail snip | |||
2. The tubes are heated at 55°C O/N | |||
3. The tubes are heated at 85°C for 45 min to inactivate the proteinase K | |||
4. After vortexing briefly, 0.5 ul of this template is added to a PCR reaction along with the other reagents: | 4. After vortexing briefly, 0.5 ul of this template is added to a PCR reaction along with the other reagents: | ||
| Line 25: | Line 24: | ||
5. PCR is then performed with the following parameters: | 5. PCR is then performed with the following parameters: | ||
94°C 3 min (initial denaturation) | |||
94°C 0.5 min (cycle*35 denature) | |||
55°C 1 min (cycle*35 anneal) | |||
72°C 1 min (cycle *35 extend) | |||
72°C 1 min (final extension) | |||
Revision as of 11:12, 21 September 2012
This protocol makes use of the DirectPCR mouse tail lysis reagent (Viagen, cat 102-T) to obtain template and the hot start Platinum Taq (Invitrogen, cat 10966-026). 1 mm tail ends are collected from mice (typically at 4-6 weeks) and the tail is degraded using the DirectPCR reagent before use in PCR.
1. 125 ul of DirectPCR reagent and 4.73 ul of Proteinase K are added to each tube containing a 1 mm tail snip
2. The tubes are heated at 55°C O/N
3. The tubes are heated at 85°C for 45 min to inactivate the proteinase K
4. After vortexing briefly, 0.5 ul of this template is added to a PCR reaction along with the other reagents:
FOR EACH PCR REACTION: 2.5 ul 10X PCR buffer 0.5 ul 10 uM dNTPs 0.75 ul 50 mM MgCl2 1 ul primer 74B* 1 ul primer 73B* 0.5 ul template (from DirectPCR) 0.1 ul Platinum Taq 17.75 ul sterile water -------- 25 ul *primer 73/74B are mixed together, so add 2 ul of this mix
5. PCR is then performed with the following parameters:
94°C 3 min (initial denaturation) 94°C 0.5 min (cycle*35 denature) 55°C 1 min (cycle*35 anneal) 72°C 1 min (cycle *35 extend) 72°C 1 min (final extension)
