IGEM:UC Berkeley/2006/PCRPrep

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Current revision (12:32, 13 September 2006) (view source)
(Culture PCR)
 
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'''******Oligos are stored at 100 uM: dilute them to 10 uM*******'''
'''******Oligos are stored at 100 uM: dilute them to 10 uM*******'''
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'''Colony PCR'''
+
==Colony PCR==
#Create PCR mixture.
#Create PCR mixture.
-
#*21.8uL
+
#*21.8uL Water
#*2.5uL or 10X thermol pol buffer
#*2.5uL or 10X thermol pol buffer
#*0.5uL dNTPs (10mM)
#*0.5uL dNTPs (10mM)
Line 14: Line 14:
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'''PCR Prep (Extend Kit)'''
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==Culture PCR==
 +
#Grow colonies in 96 well blocks overnight
 +
#Spin the block in a swinging bucket centrifuge (full speed~ 3500rpm, 5-10 minutes) to pellet the cells
 +
#Flick out the media
 +
#Resuspend the cells in the original media volume of water
 +
#Create PCR mixture.  Multiply each of the volumes below x the number of samples plus 1.
 +
#*20.8uL Water
 +
#*2.5uL or 10X thermol pol buffer
 +
#*0.5uL dNTPs (10mM)
 +
#*0.1uL Taq
 +
#*0.5uL Forward primer (10uM)
 +
#*0.5uL Reverse primer (10uM)
 +
 
 +
#Add 24 uL of mixture to each PCR tube.
 +
#Add 1uL of resuspended cells to each PCR tube
 +
#On the PCR machine use the "JED Colony" program and change the 72C cycle such that it matches the time needed to transcribe your product.  Taq goes at about 1kb/minute.
 +
 
 +
==PCR Prep (Extend Kit)==
# 36.25 ul water
# 36.25 ul water
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'''PCR Prep (Phusion Kit)'''
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==PCR Prep (Phusion Kit)==
#Create PCR mixture
#Create PCR mixture
-
#35.5 H2O
+
#*35.5 H2O
#*10 uL 5x Buffer
#*10 uL 5x Buffer
#*1uL dNTP (10mM)
#*1uL dNTP (10mM)

Current revision

******Oligos are stored at 100 uM: dilute them to 10 uM*******

Contents

Colony PCR

  1. Create PCR mixture.
    • 21.8uL Water
    • 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.
  4. On the PCR machine use the "JED Colony" program and change the 72C cycle such that it matches the time needed to transcribe your product. Taq goes at about 1kb/minute.


Culture PCR

  1. Grow colonies in 96 well blocks overnight
  2. Spin the block in a swinging bucket centrifuge (full speed~ 3500rpm, 5-10 minutes) to pellet the cells
  3. Flick out the media
  4. Resuspend the cells in the original media volume of water
  5. Create PCR mixture. Multiply each of the volumes below x the number of samples plus 1.
    • 20.8uL Water
    • 2.5uL or 10X thermol pol buffer
    • 0.5uL dNTPs (10mM)
    • 0.1uL Taq
    • 0.5uL Forward primer (10uM)
    • 0.5uL Reverse primer (10uM)
  1. Add 24 uL of mixture to each PCR tube.
  2. Add 1uL of resuspended cells to each PCR tube
  3. On the PCR machine use the "JED Colony" program and change the 72C cycle such that it matches the time needed to transcribe your product. Taq goes at about 1kb/minute.

PCR Prep (Extend Kit)

  1. 36.25 ul water
  2. 5 ul Expand tube "2" buffer
  3. 5 ul dNTP
  4. 1 ul 10 uM oligo1
  5. 1 ul 10 uM oligo2
  6. 1 ul DNA template
  7. .75 ul Expand tube "1" polymerase

50 ul total volume


Expand reagents are in Chris Anderson's -20 stuff
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
    • 35.5 H2O
    • 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

Phusion polymerase adds at 1kbp/30sec so adjust 72C step accordingly. Also, if using the program 'JEDCOLONY' there is no need for a lysis step if you are PCRing from a miniprep. The 5min 92C step can be eliminated of shortened significantly.

Keep polymerase on ice at all times

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