Ligation SOE PCR with pJH1

3/30/2015
ClpX, ClpB (insert)
pJH1 ,Δ dephosphatase and Δ SmaI (vector)
ClpX Insert 3 μL : pJH1 1 μL + 6 dH2O = total 10  μL 
ClpB Insert 3 μL : pJH1 1 μL+ 6 dH2O = total 10  μL  , brief centerfugation 

Ligation using .neb.com/protocols

Quick Ligation Protocol (M2200)

Buffers

Quick Ligation Reaction Buffer

Protocol

1. Combine 50 ng of vector with a 3-fold molar excess of insert. Use NEBioCalculator to calculate molar ratios. Adjust volume to 10 μl with dH₂O.
2. Add 10 μl of 2X Quick Ligation Buffer and mix.
3. Add 1μl of Quick T4 DNA Ligase and mix thoroughly.
4. Centrifuge briefly and incubate at room temperature (25°C) for 5 minutes.
5. Chill on ice, then transform or store at -20°C.
6. Do not heat inactivate. Heat inactivation dramatically reduces transformation efficiency.