Biology Reference
In-Depth Information
3. Digest 3
g of the shuttle plasmid with two appropriate
restriction enzymes. Use 10 U of each enzyme and allow the
digestion to proceed overnight (
see
Note 2
).
4. Confi rm complete digestion by 1 % agarose gel electrophore-
sis. Purify the DNA fragment containing the expression cas-
sette using a commercial DNA fragment purifi cation kit.
5. Quantify the recovered DNA by measuring absorbance at
260 nm.
6. Mix gently 50 ng of linearized pKP1.4 plasmid with different
amounts of the purifi ed DNA fragment (
see
Note 3
). Start
with the following molar ratios:
1:5 pKP:fragment (approx. 50 ng pKP: 50 ng of fragment) or
1:20 pKP:fragment (approx. 50 ng pKP: 200 ng of fragment).
μ
7. Transform competent BJ5183
E. coli
bacteria, using standard
procedures.
8. Plate co-transformed bacteria in LB + Ampicillin dishes.
Incubate overnight at 37 °C.
9. Pick at least 12 isolated small colonies. Inoculate each in 3 mL
of LB + Ampicillin. Incubate overnight at 37 °C with shaking
at 220 rpm (
see
Note 4
).
10. Purify plasmid DNA with a commercial DNA minipreparation
kit. Resuspend DNA in 30
l of Milli-Q H
2
O. Check by
agarose gel electrophoresis (1 %). Store at −20 °C (
see
Notes 5
and
6
).
11. Transform competent
E. coli
(strain TOP10, DH5
μ
α
or
similar).
12. Culture co-transformed bacteria in LB + Ampicillin dishes.
Incubate overnight at 37 °C. Pick two colonies from each plate
presenting less than 1,000 colonies and inoculate each in 3 mL
LB + Ampicillin. Grow overnight at 37 °C with shaking at
220 rpm.
13. Purify plasmid DNA with a commercial DNA minipreparation
kit and store at −20 °C.
14. Proceed to check and identify positive recombinants with
restriction enzymes (Subheading
3.1.2
).
Integrity and identity of the vector genome can be quickly ana-
lyzed by restriction enzyme digestion. Since each gene of interest
has a specifi c DNA sequence, a set of informative restriction
enzymes must be previously chosen by comparing with a sequence
analysis program the expected recombinant adenovirus to the orig-
inal backbone plasmid.
3.1.2 Identity
of the Vector Genome
by Restriction Enzyme
Analysis
