PCR Protocol for Microarray Chip Printing

Bradfield Lab Revised 9/14/00

Supplies:

• Sterile nanopure

• dNTP’s (100mM)

• Sense and Antisense M13 Primers 100pM/ul (ol2098 and ol2099)

• Taq (Leave in freezer until ready to use)

• 10x PCR Buffer w/ MgCl2 from Promega

• 4 Sterile 15ml falcon tubes

• Template bacterial stocks

• 4 96-well PCR plates

• 4 New Sealing Mats

Reagents:

• Primer Working solutions: Dilute each oligo 1:10 for a final concentration of 10pM/ul.

• dNTP working solution: Combine equal amounts of each dNTP for a 25mM solution. Dilute this mixture 1:10 for a final working solution.

• Lysis Buffer: 813ul nanopure + 125ul 10xPCR Buffer. Make up to 15mls at a time and store at –30C.

• PCR Master Mix for 100 reactions:
  • 6200ul Nanopure
    
  • 1000ul 10x PCR Buffer w/MgCl2
    
  • 200ul dNTP working solution
    
  • 200ul sense primer (1:10)
    
  • 200ul antisense primer (1:10)
    
  • 200ul Taq (add when ready to aliquot)
    

Protocol

1. Remove 384-well glycerol stock plate from -80C freezer. Remove tape from plate and thaw at room temperature or colder.

2. Add 6ml of Lysis Buffer to a sterile reagent reservoir. Using a multichannel pipette distribute 15ul to each well of the PCR plates.

3. Add 5ul of bacterial glycerol stock to the lysis solution.

4. Run Lysis program on thermocycler 95C/15min-> 4C/Hold

5. While cycling, prepare PCR Master Mix. Add Taq just as lysis products are ready to be removed from the thermocycler.

6. Aliquot PCR Master Mix into a sterile reagent reservoir.

7. Using multichannel pipette add 80ul of PCR master mix to each well of the lysis plate.

8. Cover with fresh sealing mat and run PCR program on thermocycler.

[95C/10sec->55C/1min->72/min] x 35 -> 4C/Hold

9. After completion of cycling, remove 5ul for amplification analysis. Freeze plates at –80C or proceed to PCR cleanup.