Transcription of 制限酵素を用いたプラスミド DNA の切断→Ligation …
1 DNA Ligation Transformation DNA pMYs(vector) 150ng/ul luc COP GFP Neo(insert) 100ng/ul Solution total250ul pMYs DNA Solution 133ul DNA20ug R4 Buffer 25ul total 1/10 XhoI 5ul DW 87ul ---------------------------------------- total 250ul luc COP GFP Neo DNA Solution 200ul DNA20ug R4 Buffer 25ul total 1/10 XhoI 5ul DW 20ul -------------------------------------- total 250ul 1.
2 Total 1/10 2. Buffer DW DNA Buffer Solution 37 C 1 10 2 1 ApE Digestion Circular 1 UV 5ul DNA5ul+LD3ul+DW2ul=10ul DNA Solution Vector BamHI 5ul Insert BamHI ScaI-HF AlwNI 5ul 37 C 1 10
3 DNA vector insert250ul 125ul 125ul Solution LD12ul Solution 30ul 4well 8well TAE buffer UV 5 10mm 2 4 GB buffer 700ul 65 C GB 1 1 1ml GB-gel Solution 2 1 700ul 2 300ul 2 4 C 15.
4 000rpm 1 GB Buffer 500ul NW buffer 700ul 2 NW buffer 1 EB Buffer 55ul 1 1 2 1 1 2 DNA DNA Ligation negative control 2 3 insert vector 2 3 TaKaRa Ligation Kit I (Enzyme Solution) 5ul 16 C 1 transformation competent cell XL-1 Blue Ligation 80 C XL-1 Blue competent cell super coil competent cell 90ul Ligation Solution 1/10 10 37 C 2 5 Cell-Ligation Solution 10 SOC 37
5 C 3 50ml 1ml Cell-Ligation Solution 37 C 2 3 150rpm 3 50ml 8,000rpm 3 100 150ul LB 37 C 1 %3192 XhoI5990 XhoI 3192 XhoI 3192 %3582 XhoI125047 XhoI 3582 XhoI 3582 100 %1926 BamHI3192 XhoI124724 XhoI 3192 BamHI 1926 791266 BamHI 1926 XhoI 3192 %550 ScaI1236 BamHI3582 XhoI4684 AlwNI1102 XhoI 3582 AlwNI 4684 22913 AlwNI 4684 ScaI 550 18686 ScaI 550 BamHI 1236 142346 BamHI 1236 XhoI 3582 46 plasmidDNA tube1 tube2 tube3 arm
6 1 l 1 l 1 l (DW) insert 4 l (DW) 4 l 4 l
