22 Expression in Eukaryotic Cells: Gene Transfer U sing Retroviral V ectors
341
Procedure
1. Use 0.025 pmol vector DNA-ends per ligation 01-!g of a 2.5 kb DNA corresponds approximatelyto 0.025 pmol DNA ends), dilute the vector DNA
to 0.025 pmol ends in 2 1-11.
2. Dilute the insert to 0.025 pmol ends in 1 1-11.
3. Set up five ligations (A - E) with the volumes (in 11l) given in the table:
A
Vector 0.0125 pmol ends/ml
2
10x ligase buffer
2
Ligase
Insert 0.025 pmol ends/ml
2
H20
13
Total Volume
20
Insert/V ector ratio
2
B
2
2
6
9
20
c
2
2
12
3
20
6
12
4. Incubate 2 hours at room temperature.
Subprotocol 4
D
2
2
15
20
Transfeetion of E. coli HB101 and Plasmid DNA Isolation
Materials
• Competent E. coli HB101 (e.g. Gibco/BRL)
• Ligation mixtures A - E
• LB media
• LB agar
• Ampicillin
• TE
E
2
12
5
20