Target Discovery and Validation Reviews and Protocols

76 Beaty et al.
The Following Volumes Are in Microliters
High mol. wt. Low mol. wt. No ligase
Fraction fraction fraction Vector only added
Vector 1 1 1 1
5X Ligase buffer 2 2 2 2
T4 Ligase (5 U/µL) 1 1 1 0
dH 2 O 0 0 6 7
Concatemers 6 6 0 0
2. Remove 1 µL of the digestion to electrophorese on a 1% agarose TAE gel with
markers, and uncut pZero. pZero should migrate in the gel at approx 2.5 kbp.
3. Add 30 µL LoTE to the rest of the sample and heat it at 70°C for 10 min to
inactivate the enzyme. The concentration is now 25 ng/L, and 1 µL can be used
per ligation.
4. Centrifuge the concatemers and pZero at 11,000g for 30 min at 4°C.
5. Wash the concatemers and pZero twice with 200 µL of 75% EtOH.
6. Resuspend each tube of the purified concatemer DNA in 6 µL of LoTE.
7. Resuspend the pZero in 30 µL of LoTE (~30 ng/L).
8. Incubate overnight at 16°C in 1.5-mL Eppendorf tubes. Before putting the ligation
in, be sure that you can continue the next day.
3.2.13. Electroporation of Ligation Products and Colony PCR
1. Add 190 µL of LoTE to the ligation mix to bring the sample volume to 200 µL.
Phenol:chloroform extract and precipitate at –80°C, and then centrifuge.
2. Transfer the upper aqueous upper layer to a new tube. EtOH precipitate the aqueous
phase in 1.5-mL tubes at –80°C.
3. Centrifuge at 11,000g at 4°C for 30 min.
4. Wash four times with 200 µL of 70% EtOH.
5. Centrifuge at 11,000g for 5 min at 4°C.
6. Remove EtOH and air-dry the pellets.
7. Resuspend each pellet in 10 µL LoTE: these mixtures are your ligation
mixtures.
8. Place ligation mixtures on ice.
9. Electroporate the 1 µL of ligation into 25 µL of ElectroMAX DH10Bs cells.
10. Plate the transformation mixture onto fresh LB-Zeocin plates (100 µg/mL
Zeocin). One hundred microliters of liquid must be dispersed on each plate.
Various amounts of both the high and low concatemer fractions are plated separately.
The cells are spread by using glass beads with sterile technique. Two dilutions
of each fraction are used: (A) 100 µL of undiluted electroporated
competent cell culture and (B) 1:10 dilution of electroporated competent cell
culture (diluted with LB media). (C) 100 µL of the two negative controls also
are plated appropriately.