1. The synthetic gene are amplified using a forward primer with
4-base-pair sequences (CACC) necessary for directional cloning on the 5
0 end of the forward primer.
2. Use a thermostable, proofreading DNA polymerase and the
PCR primers above to produce a blunt-end PCR product.
3. Check the size of the PCR product by agarose gel, using 0.8%
agarose in TAE.
Table 1
T cell antibody staining for FACS
Marker
Fluorochrome
CD25
FITC
CD3
Alexa Fluor 700
CD3
FITC
CD4
APC
CD4
PerCP-Cy5.5
CD8
PE
CD8
V500
FoxP3
PE
Antibodies against T cell membrane proteins and transcription factor. (Adapted from
Are ´valo et al. [4])
Fig. 1 Vaccine construct design and verification. (a) Schematic representation of
the vaccine construct EFn-3ÂM2e-HA2. (b) Representative verifications by
SDS-PAGE and western blot of EFn-3ÂM2e-HA2. F purification flow-through,
W wash, E eluate. (Adapted from Are ´ valo et al. [4])
428
Diana Diaz-Are ´ valo et al.
4-base-pair sequences (CACC) necessary for directional cloning on the 5
0 end of the forward primer.
2. Use a thermostable, proofreading DNA polymerase and the
PCR primers above to produce a blunt-end PCR product.
3. Check the size of the PCR product by agarose gel, using 0.8%
agarose in TAE.
Table 1
T cell antibody staining for FACS
Marker
Fluorochrome
CD25
FITC
CD3
Alexa Fluor 700
CD3
FITC
CD4
APC
CD4
PerCP-Cy5.5
CD8
PE
CD8
V500
FoxP3
PE
Antibodies against T cell membrane proteins and transcription factor. (Adapted from
Are ´valo et al. [4])
Fig. 1 Vaccine construct design and verification. (a) Schematic representation of
the vaccine construct EFn-3ÂM2e-HA2. (b) Representative verifications by
SDS-PAGE and western blot of EFn-3ÂM2e-HA2. F purification flow-through,
W wash, E eluate. (Adapted from Are ´ valo et al. [4])
428
Diana Diaz-Are ´ valo et al.