Knight:Colony PCR

From OpenWetWare

(Difference between revisions)
Jump to: navigation, search
Line 36: Line 36:
==Notes==
==Notes==
 +
#Lately, I've been carrying out this procedure using a multichannel pipettor to speed steps up.  I use a P50 multichannel pipettor to add 20μL water to the appropriate number of wells in a 96 well plate/  Picking colonies is essentially the same except that I inoculate the colony into a 96 well plate rather than a tube.  The PCR is carried out in 8 tube strips rather than individual tubes.  After adding 9.5μL of the primer + PCR supermix master mix to each PCR tube, I transfer 1μL of colony-water mixture to the reaction tube using a P10 multichannel pipettor.  (I transfer 1 μL rather than 0.5μL because it is easier to pipette.)  I then touch spin the PCR tube strips in a rack using a swinging bucket rotor in order to collect the contents of the tube to the bottom.  The PCR conditions are the same.  --[[Reshma Shetty | RS]]

Revision as of 16:16, 21 November 2005

See Colony PCR for general information about this protocol and other variants

Contents

Materials

  • Sterile 0.6mL plastic tubes
  • LB-agar plate with appropriate antibiotic
  • Oligonucleotide pair (usually VF2 and VR)
  • PCR supermix
  • PCR machine
  • Pipetman
  • Sterile pipet tips

Procedure

Picking colonies

  1. Prepare one sterile 0.6mL tube with 20μL ddH2O for each colony you intend to pick.
  2. Prepare LB-agar plate with appropriate antibiotic to use as index plate.
  3. Pick single colony using a pipetman with sterile tip. The pipettor should be set to 3μL
  4. Inoculate tip with colony into tube. Pipet up and down to ensure cells are transferred to tube. Pipet 3μL of cells suspended in water onto index plate.
  5. Repeat for as many colonies as you intend to pick.

Reaction mixture

1X Reaction

  • 9 μL PCR supermix
  • 0.25 μL 40nM VF2
  • 0.25 μL 40nM VR
  • 0.5 μL colony template

PCR conditions

  1. 95°C for 15 mins
  2. 94°C for 30 secs
  3. 56°C for 30 secs
  4. 68°C for 1 min per kb of expected product
  5. Repeat 2-4 39 times.
  6. 68°C for 20 mins
  7. 4°C forever

Run a gel to determine amplification product length.

Notes

  1. Lately, I've been carrying out this procedure using a multichannel pipettor to speed steps up. I use a P50 multichannel pipettor to add 20μL water to the appropriate number of wells in a 96 well plate/ Picking colonies is essentially the same except that I inoculate the colony into a 96 well plate rather than a tube. The PCR is carried out in 8 tube strips rather than individual tubes. After adding 9.5μL of the primer + PCR supermix master mix to each PCR tube, I transfer 1μL of colony-water mixture to the reaction tube using a P10 multichannel pipettor. (I transfer 1 μL rather than 0.5μL because it is easier to pipette.) I then touch spin the PCR tube strips in a rack using a swinging bucket rotor in order to collect the contents of the tube to the bottom. The PCR conditions are the same. -- RS
Personal tools