PrbbBB:colony pcr v1

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'''III Rescue positive clones'''
'''III Rescue positive clones'''
# inoculate positive clones from "Copy" plate for miniprep (and cell-stock) into a fresh sterile deepwell plate (preferably using a rich medium like 2xTY or 2xLB)
# inoculate positive clones from "Copy" plate for miniprep (and cell-stock) into a fresh sterile deepwell plate (preferably using a rich medium like 2xTY or 2xLB)
 +
# seal plate with gas-transmissible adhesive tape
# grow over night with vigorous shaking (700 r.p.m.) at 37°C
# grow over night with vigorous shaking (700 r.p.m.) at 37°C

Revision as of 08:06, 21 March 2009

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Contents

Overview

A colony PCR protocol for screening up to 8 clones each for up to 12 different assembly reactions in parallel. Colonies are picked into a 96-well plate with water. The PCR reactions are set up in another 96 well plate and the template DNA is copied over with a multi-channel pipette. During the colony picking, a copy of each clone is put on LB plates. This step can also be performed with a multi-channel pipette (the bugs won't survive for too long in water so it's better to do this whenever you finish a new row).

Materials

  • two sterile 96-well PCR plates
  • two sterile 96-deepwell plates (preferably 2 ml volume/square well)
  • adhesive tape for plate sealing
  • gas-transmissible adhesive tape for deepwell plate sealing
  • 12-channel 1-10µl pipette
  • multi-dispensing pipette (2 µl minimum)
  • AmpliTaq DNA Polymerase 5 U/µl (Roche)
  • AmpliTaq Buffer 10 x
  • dNTP mix 10mM each nucleotide
  • ddH2O
  • primers:
  • LB "backup" plates (with Ampicilin), one for each assembly reaction

Procedure

I PCR reaction

11µl single reaction 60xMaster 115xMaster
H2O 7.5µl 450 862
10x Amplitaq buffer 1.1µl 66 126.5
10mM dNTP 0.22µl 13.2 25.3
VF2 primer 10 µM 0.55µl 33 63.3
VR primer 10 µM 0.55µl 33 63/3
AmpliTaq 5U/µl 0.1µl 6 11.5
template DNA 1µl -- --
PCR Program
10' @95°C
30 × (30" @95°C; 15" @64; text@72°C);
optional: 10' @72°C
∞ 4°C
  • extension time text = (kb insert length) × 1' (1 min)
  1. prepare 96-well "Lysis" (PCR) plate:
    1. sterilize plate and pipette tips
    2. multi-dispense 50 µl H2O into each well
  2. prepare 96-deepwell "Copy" plate:
    1. sterilize plate and pipette tips
    2. multi-dispense 100 µl sterile LB + appropriate antibiotic into each well
  3. colony picking:
    1. pick colonies of one assembly with sterile 10 µl tips into...
    2. ... the wells of one column (like A1-H1 or A2-H2) on the Lysis plate
    3. pipette up and down
    4. eject each tip into the same position on the "Copy" deepwell plate
    5. proceed to next column for next assembly
  4. seal the "Copy" deepwell plate, shake vigorously for 5 min and let it stand at room temperature
  5. prepare PCR plate:
    1. multidispense 10 µl PCR mastermix into each well
    2. copy 1 µl per well from lysis plate to PCR plate (using multi-channel pipette)
    3. seal PCR plate with adhesive tape
  6. run PCR program

II Agarose Gel

  1. prepare a large 0.7 - 1.5 % Agarose gel (depending on fragment size)
  2. multi-dispense 2 µl loading buffer into each well of the PCR plate
  3. load gel with multi-channel pipette
  4. run, analyze, enjoy!

III Rescue positive clones

  1. inoculate positive clones from "Copy" plate for miniprep (and cell-stock) into a fresh sterile deepwell plate (preferably using a rich medium like 2xTY or 2xLB)
  2. seal plate with gas-transmissible adhesive tape
  3. grow over night with vigorous shaking (700 r.p.m.) at 37°C

Notes

Please feel free to post comments, questions, or improvements to this protocol. Happy to have your input!

raik: no comment


References

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