Protocol for Moving XO/XS Clones into an Expression Vector v.2.27.08

Materials:

Nunc Fritted Deep Well Plate
Nunc #278011

Nunc Glass Fiber 96-well Filter Plate
Nunc #278010

Eppendorf Perfectprep Plasmid 96 Vac, Direct Bind Reagents

Cre recombinase 
Clontech #63114

Acceptor vector: pMK33 CFH BD, pMK33 CHF BD, pMK33 CTAP BD, pMK33 NTAP BD, pUAST CTAP BD, pUAST NTAP BD
House

Max Efficiency DH5a Chemically Competent Cells
Invitrogen #18258-012

LB/Chlor/7.5% Sucrose
House

Sigma 10X PCR Buffer
Sigma #P2192-5ML

AmpliTaq DNA Polymerase
House

10mM each dNTP Solution
NEB #N0447S

CP1 Primer 20uM
5'-CGTCTTTCATTGCCATACGA-3'
Invitrogen

PP1 Primer 20uM
5'-TCCGCTCATGAGACAATAACC-3'
Invitrogen

Inoculate XO or XS pDONR clones:

  1. Inoculate 5uL of cells from a stock XO or XS plate into 1mL of 2xYT/Amp
  2. Place the growth block in a 37°C incubator/shaker shaking at 300RPM for 18 hours

DNA prep:

  1. Proceed with the Eppendorf Perfectprep Plasmid 96 Vac, Direct Bind DNA prep protocol outlined in Eppendor's manual with the following exceptions:
  2. Exception #1: In place of Eppendorf;s Filter Plate A, use Nunc's Fritted Deep Well Plate
  3. Exception #2: In place of Eppendorf's Filter Plate DB, use Nunc's Glass Fiber 96-well Filter Plate
  4. NOTE: The height of the Nunc filters are approximately 3mm taller than the Eppendorf filters and therefore the microtiter plate used as an adapter to support Filter Plate DB will be too tall for the Glass Fiber filter plate. Use a folded stack of paper towels instead.
  5. Elute the DNA with 60uL of ddH2O as usual.
  6. Take a spec reading of several wells (e.g. A1, A12, H1, H12) to get an approximation of the DNA concentration for the plate..

Cre reaction:

Per well:
XO or XS Donor clone (100-400ng) 1-5uL
Acceptor vector (200ng) 1.0uL
10X Cre reaction buffer 1.0uL
10X BSA (1mg/mL) 1.0uL
Cre recombinase 0.2uL
dH2O to 10uL
10uL

Perform in a PCR machine:

25O°C 12 Minutes
72O°C 7 Minutes
4O°C ∞

Transformation

  1. Thaw Max Efficiency DH5a chemically competent cells on ice for 5-10 minutes. You will need 5 tubes of cells (~1,250uL total) per plate of 96 Cre reactions.
  2. Aliquot 13uL competent cells to each well of a skirted PCR plate chilled on ice.
  3. Add 1.3uL Cre reaction to competent cells and pipette and swirl to mix.
  4. Seal the top of the PCR plate with a foil plate sealer.
  5. Incubate the PCR plate on ice for 30 minutes.
  6. Heat shock cells in a 42°C water bath for 35 seconds.
  7. Place cells back on ice for 2 minutes.
  8. Remove the foil plate sealer and add 150uL SOC to each well.
  9. Seal the top of the PCR plate with a breathable plate sealer.
  10. Place the PCR plate in its base and put the plate in a 37°C shaking incubator at 250RPM for 1 hour.
  11. Plate the entire sample onto a LB/Chlor/7.5% sucrose plate.

Pick colonies:

  1. Pick one colony per target into 500uL 2XYT/Chlor growth media. Avoid picking small colonies.
  2. Place the growth blocks in a 37°C incubator/shaker shaking at 300RPM for 18 hours.
  3. Make a 100uL frozen stock (in 7.5% DMSO) of the overnight growth blocks.

Waterprep and sequence:

  1. Water prep and sequence 5' end with MtPRUPnew primer