Biology Reference
In-Depth Information
vector genomes per cell ( N ) for each sample using the follow-
ing formula:
Na
= 2*
/
b
a = number of vector copies in the reaction.
b = number of albumin gene copies in the reaction.
Determine the number of vector genomes per cell for each
dilution of the vector suspension and for each dilution of the refer-
ence vector. By plotting log( N ) with respect to log(dilution fac-
tor), evaluate the average shift in transduction effi ciency between
the two vector preparations. This difference in effi cacy can be used
to determine the infectivity titer of the unknown vector with
respect to the reference vector.
A Coomassie gel is performed in order to assess vector purity
(Fig. 1d ). The three denatured AAV6 capsid proteins VP1, VP2,
and VP3 should appear as clear bands with approximate molecular
weights of 82, 66, and 60 kDa, respectively. The potential presence
of nonspecifi c bands of variable molecular weights is indicative of
proteinic contaminants.
3.2.4 Vector Purity
Assessment
1. Acrylamide gel preparation . Prepare a 10 %, 1 mm acrylamide
separating gel by mixing as follows: 2.5 ml 40 % acrylamide
solution, 2.5 ml 4%SDS/Tris 1.5 M pH 8.8, 4.9 ml H 2 O,
100
l TEMED. APS and TEMED are added
last as they catalyze gel polymerization. Once they have been
added, rapidly transfer the mix to the gel cassette. Gently add
ultrapure water dropwise on top of the gel to remove bubbles.
Once the gel has polymerized, pour the water out of the cas-
sette. Prepare the stacking gel by mixing as follows: 620
μ
l 10 % APS, 10
μ
l
40 % acrylamide solution, 1.26 ml 4 % SDS/Tris 0.5 M
pH 6.8, 3.06 ml H 2 O, 50
μ
l TEMED. Pour the
solution on top of the separating gel. Let the stacking gel
polymerize for 20 min before loading.
2. Sample preparation . Denaturate a vector suspension contain-
ing 3 E 10 vg, mixed with 1× loading buffer and water in a vol-
ume accommodated by the wells, by heating for 10 min at
96 °C. Load and run the gel until the loading buffer front
migrates out of the gel.
3. Coomassie staining . Rinse the gel with 100 ml of ultrapure
water for 5 min with gentle shaking. Repeat this step two
times. The gel is then stained with 20 ml SimplyBlue SafeStain
for 1 h at room temperature on gentle shaking. Discard the
staining solution and wash the gel with 100 ml of bidistilled
water for 5 min. Discard water and proceed with a second
wash with 100 ml of bidistilled water for 1 h at room tempera-
ture on gentle shaking.
μ
l APS, 5
μ
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