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
+
#* 20 μL flash mastermix
-
## 2 μL of each primer being used
+
#* 2 μL of each primer being used
-
## ~5 ng of DNA.  
+
#* ~5 ng of DNA.  
-
## total reaction volume = The total volume  '''40 μL'''
+
#* 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.
 +
#* Our primers were designed to have an annealing temperature of ~ 65oC.
 +
#**"iGem Flash" = 35-40 cycles:
 +
##98oC - 10s
 +
##98oC - 5s
 +
##63oC - 5s
 +
##72oC - 1min
 +
##72oC - 3min
 +
##4oC - infinite
# Once the PCR is finished, run 4 μL of each sample on a diagnostic gel to visualize approximate band lengths.
# 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.
# 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.
# Purify each sample using a similar approach to the Gibson DNA purification protocol.

Latest revision as of 22:04, 15 September 2011


High-Yield PCR (Full-Gene Assembly)

  1. 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
  2. Supplement the remaining volume with water.
  3. Load the sample in the PCR machine and select an appropriate program based on the primer design.
    • Our primers were designed to have an annealing temperature of ~ 65oC.
      • "iGem Flash" = 35-40 cycles:
    1. 98oC - 10s
    2. 98oC - 5s
    3. 63oC - 5s
    4. 72oC - 1min
    5. 72oC - 3min
    6. 4oC - infinite
  4. Once the PCR is finished, run 4 μL of each sample on a diagnostic gel to visualize approximate band lengths.
  5. Incubate the remaining 36 μL of each sample with 1 μL DPN 1 (enzyme) @ 37oC for 1 hour.
  6. Purify each sample using a similar approach to the Gibson DNA purification protocol.
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