05-Exercize I
Opened: Monday, 22 November 2021, 9:00 AM
•
find
a cloning
strategy
to
subclone
the NRG1-III beta3 from
the vector
pCR-bluntII-topo OR from the vector pEGFP-C3 into the vector pCMV-Tag4
•
identify
which
restriction
sites
can be
used
to
recover
the insert
from
one
vector
and which
can be
used
to
clone the insert
•
identify
which
of
the three
plasmids
(4A, 4B or 4C) do you
have
to
use
to
maintain
the frame between
the NRG1 and the FLAG
-> Translate the protein that you obtain after the cloning:
if you have the correct frame you will obtain the NRG1 followed by the FLAG: DYKDDDDK
•
if
using
plasmid
4A protein
NRG1 is
NOT in frame with
the FLAG,
try the other two plasmids: 4B and 4C,
(you have to add A to obtain plasmid 4B or AA to obtain plasmid 4C to the multiple cloning site where there is an asterisk *)
• prepare a sequence containing the
multiple cloning site of the vector and NRG1 and, using neb-cutter, translate
it to verify that NRG1 is in frame with the FLAG
•
• in a slide show the translation of NRG1
+ flag (DYKDDDDK), with the information about the restriction enzymes you used
to clone it
• if your insert can enter either sense or
antisense, please find a restriction enzyme digestion to
determine the insert orientation and write in the slide the name of the enzyme
and the length of the fragments that you obtain when the insert is sense or
antisense
• you can up-load the slide with map and
cloning information on moodle
• Please,
up-load a single slide with all information!