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Team:Washington/Protocols/High PCR
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# In an appropriately labeled tube, combine the following: | # In an appropriately labeled tube, combine the following: | ||
| - | # | + | #* 20 μL flash mastermix |
| - | # | + | #* 2 μL of each primer being used |
| - | # | + | #* ~5 ng of DNA. |
| - | # | + | #* total reaction volume = The total reaction volume '''40 μL''' |
# Supplement the remaining volume with water. | # Supplement the remaining volume with water. | ||
# Load the sample in the PCR machine and select an appropriate program based on the primer design. | # Load the sample in the PCR machine and select an appropriate program based on the primer design. | ||
Revision as of 05:18, 14 September 2011
High-Yield PCR (Full-Gene Assembly)
- In an appropriately labeled tube, combine the following:
- 20 μL flash mastermix
- 2 μL of each primer being used
- ~5 ng of DNA.
- total reaction volume = The total reaction volume 40 μL
- Supplement the remaining volume with water.
- Load the sample in the PCR machine and select an appropriate program based on the primer design.
- Once the PCR is finished, run 4 μL of each sample on a diagnostic gel to visualize approximate band lengths.
- Incubate the remaining 36 μL of each sample with 1 μL DPN 1 (enzyme) @ 37oC for 1 hour.
- Purify each sample using a similar approach to the Gibson DNA purification protocol.
