Biomedical Engineering Reference
In-Depth Information
3 Mix diluted DNA and diluted lipid gently together and incubate for 5min at room
temperature.
k
4 Incubate for a further 20 min at room temperature, before adding 1.2 ml culture medium
(no supplements). Complexes are ready for assessing (as described in Section 11.2.5) or
adding to cells.
Notes
h
Lipofectamine 2000 can be used to deliver any DNA expression plasmid. Begin by using a
plasmid containing a reporter gene (see Section 11.2.8).
i
Prepare the complexes in the same culture medium used to grow the target cells, but without
any added supplements (i.e. no serum).
j
Volume of complexes prepared for samples in triplicate (i.e. 1.5ml total, 0.5ml per well). DNA
used at 10
μ
g/ml with a weight : weight ratio of 2 : 1 for lipid-DNA complexes.
k
Do not vortex.
PROTOCOL 11.6 Preparation of Peptide-DNA Complexes
Equipment and reagents
Peptide at 1 mg/ml in sterile PBS
l
•
Endotoxin-free plasmid DNA at 1mg/ml in water
m
•
Culture medium without supplements
n
•
Vortex mixer.
•
Method
1Dilute15
μ
l DNA with 1455
μ
l culture medium (no supplements).
o
2 Add 30
μ
l peptide dropwise whilst vortexing the dilute DNA solution.
3 Vortex solution for a further 10 s.
4 Incubate the complexes for 30min before assessing (as described in Section 11.2.5) or
adding to cells.
Notes
l
This method is applicable to any synthetic peptide to be tested as a non-viral vector.
m
Any DNA expression plasmid can be used. Begin by using a plasmid containing a reporter gene
(see Section 11.2.8).
n
Prepare the complexes in the same culture medium used to grow the target cells, but without
any added supplements (i.e. no serum).
o
Volume of complexes prepared for samples in triplicate (i.e. 1.5 ml total, 0.5 ml per well). DNA
used at 10
μ
g/ml with a weight : weight ratio of 2 : 1 for peptide-DNA complexes.
