Retroviral Delivery of ECM Genes to Cells - Methods in Molecular Biology: Extracellular Matrix Protocols - page 194

4. Set up the ligation reaction. Calculate the amount of vector and insert needed for

ligation reaction in accordance with the size and molar ratio of vector and insert.
For example, 1:1 molar ratio (vector/insert) requires 100 ng of 5 kb vector and
20 ng of 1 kb insert.
For blunt ligation:

X µ L of vector (100 ng)

Y µ L of insert

1 µ L 5 m M ATP

1 µ L of 10X buffer

1 µ L 25% PEG

1 µ L T4 ligase

Z µ L H 2 O

10 µ L

5. Transform the ligation mixture to a competent E. coli strain according to the

manufacturer's instruction ( see Note 2 ). Isolate and purify plasmid DNA from
many bacterial colonies using Mini Prep kit and identify the desirable recombi-
nant plasmid by restriction digestion, PCR, and sequencing.

6. Perform a large-scale plasmid preparation of a recombinant retroviral vector. We

routinely use the Maxi Prep kit for plasmid preparation and found it satisfactory
for transfection.

3.2. Preparation of Packaging Cells
A detailed description of NX cells is available from the web site from

Dr. Nolan's laboratory (http://www-leland.stanford.edu/group/nolan/NL-
Homepage.html). The

Φ

Φ NX cells are maintained in growth media containing
DMEM, 10% FBS, 50,000 U/500 mL penicillin, 50 mg/500 mL streptomycin,
1% glutamine and splitted 1:4 or 1:5 every 3-4 d. The

NX cells are seeded at
3 million cells per 10 cm plate in growth medium, 18-24 h prior to transfec-
tion. A high-transfection efficiency is achieved when cells are confluent
40-50% at the time of transfection. Significant reduction in transfection effi-
ciency results when cells are confluent.

Φ

3.3. Transfection
1. Change the medium with a solution containing 0.1% of BSA instead of serum 3 h

prior to transfection. For the Tet-off system, add tetracycline (1-2 µ g/mL) or

doxycycline (20 ng/mL-1 µ g/mL) at this point ( see Notes ).

2. Put 0.5 mL of 2XHBS into one Eppendorf tube.

3. Into another Eppendorf tube, add H 2 O + DNA + CaCl 2 . We suggest using 2 M

CaCl 2 (62.5 µ L/mL of reaction mixture). The best results were obtained using

g of DNA/1 mL of the reaction mixture. Dilute DNA with H 2 O and add
CaCl 2 dropwise. The final volume of this mixture should be 0.5 mL.

20 µ