Biomedical Engineering Reference
In-Depth Information
3. Decant the media from the cell pellets by inverting the block
and drain by tapping onto a paper towel.
4. Seal the plates and store at −80 °C for a minimum of 20 min
before Ni
2+
-NTA screening.
3.6 Ni
2+
-NTA
Miniature Expression
Screen Protocol
This manual protocol is similar to the automated protocol used in
OPPF-UK (
Note 13
).
1. Resuspend the defrosted cells completely in 210
L of Lysis
Buffer. This cans be done either with a suitable multichannel
pipette or on an orbital shaker (~1,000 rpm for 30 min).
If resuspending using a multichannel pipette, once resuspended
incubate for 30 min at room temperature to allow for the
action of the lysozyme and DNAse I.
2. Centrifuge the deep well block at 6,000 ×
g
for 30 min at 4 °C.
3. Dispense 20
μ
L of the Ni-NTA magnetic bead suspension into
each well of a fl at-bottomed microtitre plate.
4. Transfer the supernatant from
step 1
without disturbing the
“insoluble” pellet to each well of the microtitre plate contain-
ing the Ni-NTA magnetic beads (
Note 14
).
5. Mix for 30 min at room temperature using either a microtitre
plate shaker at 600 rpm or vortex using an adapter for microtitre
plates.
6. Place the plate on the 96-well magnet once the supernatant
has clarifi ed; remove the supernatant carefully from the beads.
7. Add 200
μ
L of Wash Buffer to each well, remove from the
magnet, and shake (or vortex) for 5 min.
8. Place the plate on the 96-well magnet and once the superna-
tant has clarifi ed remove the buffer.
9. Repeat
steps 7
-
8
.
10. Add 60
μ
L of Elution Buffer (NPI-250) to each well, shake
(or vortex) for 1 min, place the plate on the 96-well magnet,
and, once it has clarifi ed, transfer the supernatant (eluate) to a
fresh plate for analysis on SDS-PAGE gels.
μ
4
Notes
1. The 5
homology extensions for cloning into the pOPIN
vectors are indicated in the vector tables. We have developed a
software tool for designing the appropriate PCR primers for clon-
ing into the pOPIN suite of vectors. This is available through the
following link:
https://www.oppf.rc-harwell.ac.uk/Opiner/
.
′
and 3
′
2. Obtaining good quality PCR products is critical. We fi nd the
easiest way to optimize PCR is to use an alternative polymerase
and would only try other variables, e.g., annealing temperature,
