Transfection Antibiotics
Suggested Protocol for Hygromycin B, Puromycin Dihydrochloride, G418 Sulfate, Blasticidin
The selection of transfectants, cells containing extrachromosomal DNA of interest, is an essential part of any cell culture
genetic manipulation. There will always be a certain number
of cells that fail to incorporate the foreign DNA, so specific
antibiotics may be used to “select” for only transfected cells.
 Step 3: Remove the growth media from the cells, maintaining aseptic conditions. Using a pipettor may work best.
Selection antibiotics enable the transfection of vectors
carrying the respective antibiotic resistance gene into the cell
along with foreign DNA. When this antibiotic is added to the
culture media after transfection, only those cells expressing
resistance to that antibiotic will survive. We offers an array
of popular selection antibiotics, including the aminoglycosides G418, Hygromycin B, Puromycin Dihydrochloride, and
Blasticidin.
 Step 5: Apply the antibiotic to the respective wells,
leaving one set of rows empty. To these wells, add growth
medium not containing the antibiotic. These wells can be
used to compare the cells under antibiotic influence and
determine the maximum effectiveness and signal the end of
the test.
Cell Preparation
 Step 7: Optimal effectiveness should be reached in 2-10
days, depending on the antibiotic. This can be seen by
almost total obliteration of cells in the highest concentrations, and little to no effect in the lower concentrations.
Note the time when the cells reach their greatest confluency.
This is the point when the cells expand to optimally cover all
areas of the well before death and flotation occur as a result of
entering the stationary phase and/or overgrowth.
 Step 6: Return the plate to the proper growth conditions
and observe daily, noting results.
 Step 8: Once the maximum effect has been reached, the
cells can be detached and stained using methylene blue and
the optical density measured.
 Step 9: If the concentration range used in the study is
broad enough, the optical densities can be graphed,
resulting in a somewhat linear curve.
The best way to do this may be to set a linear concentration of
cells/well and measure the absorbance daily. This will
allow measurement of concentration/well and to determine
the best seed volume and growth time to confluence. Cells
must still be in log phase to allow for uptake of the antibiotic
and resulting response.
 Step 10: Several curves may need to be performed to
determine the concentration that delivers the best effect to
the cells without causing adverse reactions.
Antibiotic Preparation
 Step 12: This dose response curve should be performed
for each new lot of antibiotic because each lot may vary in
purity or potency. Cell conditions may also change.
Dilute the antibiotic to a broad linear concentration range using the appropriate growth medium as the diluent. The wider
the concentration range, the better assessment of
optimal user concentration.
Procedure: Performing Antibiotic Dose-Response Curves
 Step 1: Set cells in the predetermined concentration and
allow them to grow to near confluence (~70-80%) under
normal growth conditions (temperature, CO2, etc.).
 Step 2: Once the target confluency is reached, dilute the
antibiotic to concentrations for testing.
 Step 11: From the data obtained in the dose-response
curve, extrapolate the appropriate concentration of
antibiotic to use to obtain the desired effect.
Cat. No.
Description
Size
Qty/Pk
30-240-CR
Hygromycin B solution
20 mL
1
61-385-RA
Puromycin Dihydrochloride, powder
100 mg
1
30-234-CR
30-234-CI
G418 Sulfate, Liquid
20 mL
100 mL
1
1
61-234-RF
61-234-RG
61-234-RK
G418 Sulfate, powder
1g
5g
50 g
1
1
1
30-100-RB
Blasticidin, powder
50 mg
1
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First, determine the appropriate seeding volume for the cell
line to be used. If using a 96-well plate, a concentration curve
may need to be set in advance to determine the best volume of
cells/well to allow for growth and to prevent death from overcrowding before application of the antibiotic.
 Step 4: Label the plate(s) to reflect the concentrations of
antibiotic applied to the cells. The best maximum volume
per well of 96-well plates may be 200 µL/well.