Digestion and dephosphorylation of vector:
20 µl:
2 µl 10xrestriction buffer
2-4 µg vector
1 µl of each restriction enzyme
37C, ≥1h.
75 µl ddw
5 µl 1M Tris-HCl pH 8.5
2 µl alkaline phosphatase (AP, Boehringer)
37C, ≥1h.
(If blunt or 3'overhangs, after incubation add 1µl AP and incubate at 50C for another 1 h.)
5 µl 0.2M EDTA (pH 8)
Extract with Phenol:CHCl3 (PCA) (leave 5-10 µl aqueous phase behind).
10 µl 3M NaAc pH 6.0
1 µl 20 mg/ml glycogen
275 µl 100% EtOH
Ice ≥ 5 min
Spin 14,000 rpm, 15-30 min at 4C
Remove EtOH
Wash with 70% EtOH
Dry pellet
Dissolve at 0.1 mg/ml in Te buffer (10 mM Tris-HCl pH 8, 0.1 mM EDTA)
Preparation of insert:
PCR product:
Purify PCR product from agarose gel
To 50 µl of gel purified PCR product add:
5 µl 10x restriction buffer
1-2 µl of each restriction enzyme
37C, ≥1h.
45 µl ddw
5 µl 0.2M EDTA (pH 8)
Extract with PCA.
10 µl 3M NaAc pH 6.0
1 µl 20 mg/ml glycogen
275 µl EtOH
Ice, ≥5 min.
Spin 14,000 rpm, 15-30 min
Remove EtOH.
Wash with 70% EtOH.
Dissolve in Te buffer (e.g. 10 µl Te for a 20 µl PCR rxn)
Plasmid insert:
20 µl:
2 µl 10xrestriction buffer
2-5 µg plasmid
1 µl of each restriction enzyme
37C, ≥1h.
5 µl 5xDNA load buffer
Purify DNA fragment from agarose gel
Elute in 30 µl Te
Oligo insert:
Phosphorylate each oligo (in sepearate rxns):
10 µl:
1 µl 10xPNK
1 µl 50 mM DTT
200 pmole DNA oligo
2 µl 2 mM ATP
1 µl Polynucleotide kinase
37C, ≥1h.
Anneal by mixing the two phosphorylation rxns
95C, 2 min
RT, 5 min
Ice.
This will give an annealed oligo concentration of 10 µM.
Use 0.001-0.1 pmole for a ligation. The amount has to be titrated every time as too high an oligo concentration results in multiple inserts.
Ligation:
10 µl:
1 µl 10xT4 DL
1 µl 50 mM DTT
1 µl 2 mM ATP
1 µl cut, cipped vector (≈0.1 mg/ml)
2-5 µl cut insert/PCR product or 0.001-0.1 pmole annealed, phosphorylated oligos (Titrate every time!)
0.2 units T4 DNA ligase (Boehringer)
RT, ≥4h.
Transform 2.5 µl to competent E. coli DH5a cells and plate on selection medium
Store remainder of ligation at -20C.
Buffers:
Te:
10 mM Tris-HCl pH 8
0.1 mM EDTA
10xT4 DL:
660 mM Tris-HCl pH 7.5
66 mM MgCl2
10xPNK
500 mM Tris-HCl pH 7.5
100 mM MgCl2
JLA; Aug. 1, 2007