Difference between revisions of "Generating and Amplifying Adenoviral Constructs"

Revision as of 18:53, 14 September 2010

Materials

• Targetting Construct in pAdtrack vector or the like (see Preparing an Adenoviral shRNA Clone )
• PmeI restriction enzyme (or other enzyme as required for linearization.
• PacI restriction enzyme
• Electrocompetent BJ5183-AD-1 cells (Agilent Cat#200157; or prepare yourself)
• 7.5 M ammonium acetate
• 20mg/mL glycogen
• 25:24:1 phenol/chloroform/isoamyl alcohol, pH 8.0
• 100% ethanol
• 2 mm electroporation cuvette

Protocol

see Nature Protocols, Current Protocols in Human Genetics protocols.

Linearization and Purification of Shuttle DNA

1. Digest 0.2-0.5 ug plasmid DNA in 100 uL volume with 30-100U of PmeI overnight at 37C. If desired check a small amount by electrophoresis to ensure complete ligation.
2. Add 100 uL ddH2O, 100 uL 7.5 M ammonium acetate and 2 uL 20 mg/ml glycogen.
3. Add 300 uL 25:24:1 phenol/chloroform/isoamyl alcohol, pH 8.0.
4. Remove top layer to clean tube and add 600 uL of 100% ethanol
5. Centrifuge 5 min at 13 000 RPM
6. Wash three times with 70% ethanol to remove residual salt.
7. Dry and resuspend in 8 uL

Transformation into BJ5183-AD-1 cells

1. Add 8 uL resuspended linearized plasmid to 20 uL electrocompetent cells
2. Carefully transfer to ice cold 2 mm electroporation cuvette avoiding bubbles and keeping on ice
3. Electroporate at pulse at 2,500 V, 200 O and 25 mF
4. Resuspend in 500 uL LB and plate on 2-5 LB/Km plates
5. Pick 10-20 of the smallest colonies the next day and grow in LB/Km broth
6. Miniprep clones. Digest 10 uL with PacI and check size by running out both digested and undigested clones on a 0.8% agarose gel. Correct recombinants usually yield a large fragment (approximately 30 kb) and a smaller fragment of 3.0 or 4.5 kb.
7. Retransform and amplify correct clone(s).