Proper percentage gel to use for different DNA sizes
Agarose
Gel Percentage
|
Size range of
DNA in base pairs
|
0.8
|
1000-10000
|
1.0
|
600-8000
|
1.3
|
300-800
|
1.5
|
150-600
|
2.0
|
75-150
|
If your DNA is smaller than 75bp or you need to distinguish
small differences between products, use a non-denaturing polyacrylamide gel of
8-10% - you can estimate migration by the fact that the xylene cyanol will migrate
at about 50bp, while the bromophenol blue migrates at around 150bp. Don’t forget, ALWAYS make fresh ammonium
persulfate when pouring your gel!
MDA PCA 2B Cell Line
Care - read this BEFORE you kill them!
When graduate student Lindsey Kelly rotated through the lab, she told me she wanted to learn how to do difficult cell culture. We gave her the most tedious line to take care of that has ever been developed; we love MDA PCA 2B cells because they express PSMA at exceedingly high levels, but growing them has always been difficult. Lindsey succeeded in not only growing these cells well, but transducing them with lenti constructs, and successfully knocking down target genes. She kindly put together this primer for growing them.
Media
HPCI
20% FBS
0.1%
Pen/Strep
0.1% L-Glut
General Cell Growth
When healthy, the cells can grow
quickly and tend to grow in little mounds when they start to become
confluent. When there are very few
cells, they are unhealthy or they have sat in the same flask too long they seem
to form dense clumps. The clumps can be
attached well or very loosely and sometimes even come off. If there are a lot of clumps, they can be
moved to a 15ml conical to be trypsinized because usually there are some cells
alive.
Conditioned media can be used on
these cells. If there is a flask of
healthy growing cells, save the media and add to new flask at 20%. Alternatively, when changing media, leave 20%
in flask and add 80% fresh media. To be
“conditioned media” it should be on cells for 3 days. Pre-warm the media in the conical if aliquoted
or in the flasks if splitting cells in a water bath or incubator.
MDA PCA 2B cells are grown on poly-L-lys treated flasks. Some will possibly grow on untreated flasks. See protocol for poly-l-lys treatment.
MDA PCA 2B cells are grown on poly-L-lys treated flasks. Some will possibly grow on untreated flasks. See protocol for poly-l-lys treatment.
Splitting Cells
1.
Remove media
a.
If cells are
loosely attached, remove media with serological pipette
b.
If cells are growing well and do not need
conserved media, aspirate media
c.
To conserve, remove media with serological
pipette and reserve in conical
2.
Add Trypsin
a.
2mL for t25, 3mL for t75
3.
Incubate at 37°C for 10 min in the incubator
4.
NO NOT tap, hit or shake or cells may clump
5.
If cells are unhealthy pipette media into
poly-l-lys treated flasks and pre-warm the media
6.
Use media to neutralize trypsin and to wash the
cells off the bottom of the flask
a.
4mL for t25, 6mL for t75
b.
Pipette up and down very gently if cells are
sickly
c.
Wash the corners a few times
7.
Move to conical and spin at .7 for 3-5 min
8.
Carefully aspirate off media from pellet
9.
Gently resuspend pellet in media and plate cells
onto poly-l-lys treated flasks
Cell Maintenance
1.
Feed pretty frequently or they start to ball up,
probably best not to go more than 4 days
2.
Also like to be moved frequently to keep from
balling up (although I’m not sure of the significance of the balls), probably
move to a fresh poly-l-lys flask weekly (even if they are not confluent enough
to be split)
3.
Healthy cells
a.
Should have some small mounds and lots of single
layer cells? I’m not sure how “bad” the mounds or the balls are
b.
Split 1:2 when close to confluent, I have never
split them more that that, but they may be fine
4.
Slow growing cells
a.
If the cells are just growing slowly, or perhaps
they are plated at a low density to begin with and then are not confluent with
in a week, move them (split 1:1)
b.
They seem to grow faster when moved to another
poly-l-lys treated plate
c.
This works well if they are just in balls or
mounds, separating them will help them to grow more
5.
Balled up cells
a.
If there are loosely attacked or floating balls
of cells, remove with a serological pipette and put in a conical
b.
Spin down to remove media and add 2mL trypsin to
cell pellet
c.
Pipette up and down and incubate for 10 min
d.
Add trypsin to plate following instructions
above
e.
Add media to the trypsin for the floating cells
in the conical to neutralize
f.
Spin all the cells, can recombine when plated or
plated separately
Transductions
1.
If using a t25 try to estimate cells to get ~30%
confluency, if cells are healthy and growing quickly, they will need to not be
overly confluent before they are put under selection
2.
There was not a lot of cell death when I did my
transductions compared to the amount of cell death with LNCaPs
3.
Use 800ng/mL of puromycin to select cells, will
take 48 hours or so to start killing the cells
4.
Keep under selection for the recommended time
 |
| healthy cells |
 |
| one large ball of cells |
 |
| clumpy cells |
Poly-L-Lysine treatment for Cell Culture flasks - save some bucks, make your own! [especially if you are in our lab...]
1.
Make a 1:10 dilution of the Poly-L-Lys and cell
culture PBS
a.
Ex: 45mL of PBS and 5mL of Poly-L-Lys
b.
Dilution can be stored in 2°-8°C for up to 3
months
2.
Dilution should be at room temp before use
3.
Add approximately 1mL/cm2 of the
Poly-L-Lys dilution to each flask
a.
~2.5mL to
t25 and ~7.5mL to t75
b.
Needs to be enough to coat the bottom of the
flask
c.
Rock to coat
4.
Incubate flasks at room temp for 30 min
5.
Label to indicate treatment and date
(‘poly-l-lys treated date’)
6.
Aspirate the Poly-L-Lys dilution (do not reuse)
7.
Rinse each flask with PBS then aspirate
8.
FOR SAME DAY USE: Incubate vented flasks at 37°C
in the incubator for 30 min
a.
Keep the flasks open (like if they had cells) so
allow some of the moisture to evaporate
b.
Some moisture will remain, the flasks are ready
to be used
9.
Store flasks tightly closed at 2°-8°C in the bag
they are shipped in
a.
After overnight storage flasks are ready to use
b.
Will see some condensation of remaining PBS
