T7Select phage cloning protocol

Vector:

T7Selct10-3b
Copy number: 5-15 proteins/phage
Size limit: 1,200 aa
Compatible host strains: BLT5403, BL5615

Bacterial strain:

BLT5403.  If there are problems, try BLT5615, as it is (magically, according to the manual) different than BLT5403.  Note that BLT5615 requires IPTG, so check the manual again before switching. 

Kit instructions for glycerol stock:

• colony from plate to 50 mL TB or LB in 250 mL flask
• @ OD600 ~ 0.6-1.0, take 1.5 mL and combine with 0.15 mL sterile 80% glycerol. 

Some recipes:

Media

Various stocks

Prepare insert:

• Amplify the insert with appropriate primers, adding EcoRI/HindIII sites on either end.
• Gel purify inserts, going into 30 uL
• Cut with EcoRI-HF/HindIII
  30 uL insert; 4 uL NEB2; 0.4 uL BSA; 2 uL EcoRI-HF; 2 uL HindIII; 1.6 uL ddH₂O
  37 °C for 2 hr.
• Purify on MinElute column into 10 uL
• Want a final concentration of 0.02-0.06 pmol/uL. 

• Linker alone is 50,950 g/mol: 1.0-3.0 ng/uL
• Linker + S100 is 160,073 g/mol: 3.2-9.6 ng/uL

Ligation:

Relatively expensive; cut volumes as small as you can go. 

• Want insert:vector ratio of 3:1.
• Recipe:
  • X uL insert (0.06 pmol total); [This is 1 uL for control]
  • 1 uL T7Select Vector Arms (0.02 pmol)
  • 2.5 uL 2x ligase buffer
  • 0.5 uL 2,000,000 U ligase
  • ddH­₂O to 5 uL
  • Pipette up and down
  • 1 hr @ room temperature; store at 4 °C

In Vitro Packaging:

• Thaw packaging extract on ice.  Subdivide into pre-chilled tubes if desired.
• Add 5 uL ligation reaction to 25 uL extract (can scale down, as long as ratio is the same).  Mix with pipette – DO NOT VORTEX. 
• For packaging positive control, add 0.5 ug control to 25 uL.
• Incubate at room temperature for 2 hr.
• Stop the reaction with 270 uL sterile LB.  To store for ~24 hr week, add 20 uL chloroform, mix gently by inversion, and store at 4 °C.  Good for up to 1 week.

Plaque Assay:

• Grow up host strain to OD600 = 1.0 in M9TB medium.  Store at 4 °C until ready for use (no more than 48 hr).
• Melt enough top agarose for dilution series (5 mL/plate) and place in 45-50 °C water bath.
• Put appropriate number of 100×15 mm LB+amp (20 mL) plates at 37 °C to prewarm.
• Make up serial phage dilutions from packaging reaction above: first pass is 10 uL + 990 uL, then 100 uL + 900 uL, etc.  Do 4 dilutions. 
• Add 250 uL host cells to 4 mL tubes. 
• Add 100 uL phage dilution to each tube.  Add 3 mL top agarose to the tube and immediately pour onto plate.  Swirl gently to spread evenly.  DO QUICKLY, as phage will begin replicating immediately. 
• Place plates right-side up until agarose solidifies, then place at 37 °C for 3-4 hr or overnight at room temperature.
• Count plaques and calculate phage titer.