Difference between revisions of "Mc4r-Cone Genotyping"
From Bridges Lab Protocols
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!| PCR Step || Temperature || Time || Cycles || Notes | !| PCR Step || Temperature || Time || Cycles || Notes | ||
|- | |- | ||
| − | 1. Initiation/Melting || 94 || 5:00 || 1 || | + | |1. Initiation/Melting || 94 || 5:00 || 1 || |
|- | |- | ||
| − | 2. Denaturation || 94 || 0:45 || 40 || | + | |2. Denaturation || 94 || 0:45 || 40 || |
|- | |- | ||
| − | 3. Annealing || 66 || 0:45 || 40 || | + | |3. Annealing || 66 || 0:45 || 40 || |
|- | |- | ||
| − | 4. Elongation || 72 || 0:45 || 40 || steps 2-4 to be run in sequence | + | |4. Elongation || 72 || 0:45 || 40 || steps 2-4 to be run in sequence |
|- | |- | ||
| − | 5. Amplification || 72 || 7:00 || 1 || | + | |5. Amplification || 72 || 7:00 || 1 || |
|- | |- | ||
| − | 6. Finish || 4 || infinite || n/a || | + | |6. Finish || 4 || infinite || n/a || |
|} | |} | ||
Revision as of 17:49, 5 June 2020
PRIMERS
- Fwd: CAC TCG GAG CTT CCC TGA CCC AG
- Rev: GAC CAT GGT TTC CGA CCC ATT
- Neo Rev: TTC CAA GCC TCT GAG CCC AGA
Make primers at 1uM (Fwd + Rev and Fwd + Neo Rev). PCR primers separately.
PCR PROTOCOL
| PCR Step | Temperature | Time | Cycles | Notes |
|---|---|---|---|---|
| 1. Initiation/Melting | 94 | 5:00 | 1 | |
| 2. Denaturation | 94 | 0:45 | 40 | |
| 3. Annealing | 66 | 0:45 | 40 | |
| 4. Elongation | 72 | 0:45 | 40 | steps 2-4 to be run in sequence |
| 5. Amplification | 72 | 7:00 | 1 | |
| 6. Finish | 4 | infinite | n/a |
