Confusion Cloning
Confusion PCR Cloning
Home made extract
[edit]
1, Grow DH10B or Top10 with pBad24-Red-alpha at 37 oC in 100 ml 2X YT medium until they reached OD600 ~ 5.3.
2. Add 0.2% L-arabinose to induce for 2 h to express lamda prophage protein Red-alpha.
3. Harvest cells by centrifugation at 4 oC.
4. Wash cells 1 time with 200 ml ddH2O and resuspend cells in 1.2 ml CelLytic B Cell Lysis Reagent (Sigma) or add lysozyme.
5. Incubate the resuspended cells at room temperature for 10 minutes to allow lysis to occur.
6. Spin cell lysates at 12000 rpm for 10 min at 4 oC to pellet the insoluble material. Collect the resulting supernatant.
7. Mix cell extracts with equal volume of 100% glycerol,
8. Aliquote into 40–60 ul per tube, store at -20o C or -80 oC for long-term storage
Confusion reaction:
[edit]50–200 ng linear vector
appropriate amount of insert DNA in a 1:1 to 10:1 molar ratio of insert to vector
1 ul 10X Confusion buffer (500 mM Tris–HCl, pH 7.5 at 25 oC, 100 mM MgCl2, 10 mM ATP, 10 mM DTT)
1 ul Confusion extract
ddH2O to a total volume of 10 ul.
Incubate at 37 oC for 1 h.
Transform homemade Top10 competent cells
In-Fusion PCR Cloning: Clontech
Cold Fusion Cloning Kit: SBI
Fast Seamless Cloning Kit: Dogene
CloneEZ Kit: Genescript
GENEART Seamless Cloning and Assembly Kit: Invitrogen
SLIC uses the 3' ->50 exo-nuclease activity of T4 DNA polymerase to generate ssDNA overhangs in insert and vector which are required for the fusion of vector and insert fragments by single strand annealing with or without the addition of
RecA.
Exonuclease III induced ligase-free directional subcloning uses 3' ->5' exonuclease activity of Exonuclease III to generate ssDNA overhangs which facilitate cloning.
Ribocloning uses Rnase A to cleave at single rC or rU bases that were introduced by PCR into vector and insert and subsequent heating to generate ssDNA overhangs for cloning.
Enzyme-free cloning creates complementary ssDNA overhangs by PCR with tailed primer sets and post-PCR denaturation-hybridization reactions.
In-Fusion PCR Cloning promotes PCR cloning by the In-Fusion enzyme, a poxvirus DNA polymerase with 3' ->5'exonuclease activity.