IGEM:UC Berkeley/2006/PCRPrep

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Line 3: Line 3:
#*21.8uL
#*21.8uL
#*2.5uL or 10X thermol pol buffer
#*2.5uL or 10X thermol pol buffer
-
#*0.5uL dNTPs (10uM)
+
#*0.5uL dNTPs (10mM)
#*0.1uL Taq
#*0.1uL Taq
#*0.5uL Forward primer (10uM)
#*0.5uL Forward primer (10uM)
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-
'''PCR Prep'''
+
'''PCR Prep (Extend Kit)'''
# 38.5 ul water
# 38.5 ul water
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-DLK_062106
-DLK_062106
 +
 +
 +
'''PCR Prep (Phusion Kit)'''
 +
 +
#Create PCR mixture
 +
#*10 uL 5x Buffer
 +
#*1uL dNTP (10mM)
 +
#*0.5uL Phusions (polymerase)
 +
#*1uL oligo 1 (10uL)
 +
#*1uL oligo 2 (10uL)
 +
#*1uL DNA Template
 +
#Run in PCR machine
 +
 +
Keep polymerase on ice at all times

Revision as of 13:33, 29 June 2006

Colony PCR

  1. Create PCR mixture.
    • 21.8uL
    • 2.5uL or 10X thermol pol buffer
    • 0.5uL dNTPs (10mM)
    • 0.1uL Taq
    • 0.5uL Forward primer (10uM)
    • 0.5uL Reverse primer (10uM)
  2. Add mixture to PCR tube.
  3. Pick colony and add to tube and then streak an "X" on a plate while making a map of which colony is where.


PCR Prep (Extend Kit)

  1. 38.5 ul water
  2. 5 ul buffer2
  3. 5 ul dNTP
  4. 1 ul oligo1
  5. 1 ul oligo2
  6. 1 ul DNA template
  7. .75 ul polymerase

50 ul total volume

Combine in PCR tube.

Notes:

To make 1ul oligo at correct concentration, take 1ul oligo and 9ul water, combine, transfer 1ul of solution to PCR tube

Keep polymerase on ice at all times

39 ul water can be used in place of 38.5ul water

-DLK_062106


PCR Prep (Phusion Kit)

  1. Create PCR mixture
    • 10 uL 5x Buffer
    • 1uL dNTP (10mM)
    • 0.5uL Phusions (polymerase)
    • 1uL oligo 1 (10uL)
    • 1uL oligo 2 (10uL)
    • 1uL DNA Template
  2. Run in PCR machine

Keep polymerase on ice at all times

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