AMGEN BIOTECH EXPERIENCE
20XSB Dilution and Agarose Gel Preparation
1XSB Buffer Preparation
The Sodium Borate electrophoresis solution is sent to you at higher concentration (stock concentration) than the concentration students actually use
(working concentration). To calculate the dilution from a stock concentration to a working concentration use the following method.
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Determine how much of the working solution you will need.
Then use the formula C1V1 = C2V2 to determine the volume of stock solution (V1) you will need to dilute to prepare the final volume of working
solution (V2).
(C=concentration, V=volume)
Example. You need to prepare 1x SB buffer (electrophoresis buffer) for 6 agarose gels. Each gel needs about 30 mL of this buffer. Amgen
supplies the SB buffer, but its concentration is 20x. 30mLx 6 gels = 180 mL
Apply the formula C1V1 = C2V2
(20x)(V1) = (1x)(180mL)
(V1)= (1x) (180 mL)
20x
( V1) = 9mL
To 9.0 mL of 20x SB, add 171.0 mL of dH2O (deionized or distilled) to make 180 mL of 1x SB electrophoresis buffer.
1XSB electrophoresis buffer is needed to run the gel in the electrophoresis chamber as well as to prepare the gels. Time can be saved by preparing a
large volume of 1xSB buffer prior to making the gels using the formula above, or by using the table below.
For Total Volume of 1X SB
1000 mL 1X SB =
500 mL 1X SB =
200 mL 1X SB =
150 mL 1X SB =
Use 20X SB
50 mL 20X SB
25 mL 20X SB
10 mL 20X SB
7.5 mL 20X SB
Use Water
950 mL
475 mL
190 mL
142.5 mL
Store the diluted buffer in several 500mL containers to make it easy for students to pour the buffer into their electrophoresis chambers. This buffer can
remain in the gel chambers and be used for several classes.
Agarose Gel Preparation
For each gel casting tray, you will need approximately 30mL of a 0.8% agarose solution. Therefore, 6 gels would require 180 mL of agarose solution. To
allow for some loss due to solution staying in the flask, round up to 200mL
0.8 = x grams of agarose
100 ?mL of 1X buffer
This is the basic formula, where ? is the volume of agarose solution desired
0.8 = x grams of agarose
100 200 mL of 1X buffer
.
(200) (0.008) = 1.6 grams of agarose (this percentage is weight of agarose over volume of 1xSB buffer)
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Place 1.6 grams of agarose into a 500 mL flask. Add 200 mL of 1x SB
Cover the flask opening with plastic wrap. Use a pipette tip to poke a small hole in the plastic wrap.
Place the covered flask in a microwave. Set the microwave for 1 minute on high. ( A hot plate can also be used to melt agarose but you will
need to use a double-boiler.)
As soon as boiling starts, gently swirl the flask with a gloved hand. Continue this procedure, reducing the time on the microwave (5 ± 15
seconds), until all of the agarose has been dissolved.
o To check this, hold the flask to the light and swirl the solution. Look carefully for “lenses” of agarose crystals suspended in the
liquid. If no lenses are visible, the agarose is dissolved.
o If your students will pour the gels, transfer 30mL melted agarose into 50mL conical tubes, cap, and keep in a 60o C water bath.
When the solution has cooled to the point that you can hold the bottom of the flask without burning your hand (60oC), the solution can be
poured into the casting trays. The solution should cover about 2mm of the comb. Do not allow the agarose solution to cool to the point that it
08-06-2014
AMGEN BIOTECH EXPERIENCE
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begins to re-solidify. If it does solidify, simply reheat the solution.
o A 50mL conical tube makes it easy to measure 30mL for each tray
o Fotodyne electrophoresis trays have “gates” that must be placed in the “up” position and secured snugly using the set of washers and
screws, before pouring the melted agarose into the tray. If the Fotodyne gates have been removed or you have Bio-Rad trays, tape
the ends of the tray with masking tape. Be sure to run your finger firmly along the masking tape to insure a tight seal. If you have
OWL trays, refer to the Kit Notebook for instructions.
o If the kit has a Multi-gel Casting Tray refer to those instructions.
Gels will take about 30 minutes to solidify
Pull the comb straight out of the gel without wiggling it back and forth. This will minimize damage to the front wall of the well.
Place the gels into zip lock baggies with a small amount of 1XSB buffer. Store flat in the refrigerator until ready to use. Gels may be stored for
several days (or weeks). Keep in mind that Labs 1, 4, and 4A require gels, you will need to prepare many gels to meet the needs of the labs.
o You can use a single comb per gel and have two groups load their samples in a single row. The Bio-Rad combs have fewer “teeth”
and may require you double-comb the gel in order to get sufficient wells for two groups.
o If the students have not damaged (holes poked in the wells) their Lab 1 gels, they may be reused. Have the students bring the tray
to you, making certain that the gel does not slide off the tray, and place the gel into a Pyrex dish or cafeteria tray in which you
have 1x SB. Place all of the gels into the buffer and allow them to sit overnight. The dyes will diffuse from the gel overnight.
Remove the gels and place them in zip lock baggies for Lab 4 or 4a. Store in the refrigerator until ready to use.
When preparing agarose gels, you can use the formula above or use the table below
For Total Volume
100 mL 0.8% Agarose gel
150 mL 0.8% Agarose gel
200 mL 0.8% Agarose gel
Use Agarose
0.8 gram
1.2 gram
1.6 gram
Use 1X SB
100 mL 1X SB
150 mL 1X SB
200 mL 1X SB
100 mL 2.0% Agarose gel
200 mL 2.0% Agarose gel
2.0 gram
4.0 gram
100 mL 1X SB
200 mL 1X SB
NOTE: If you are running the PCR lab, you will need to prepare 2% agarose gels. These will be double combed with the small toothed combs. Since you
use one student sample per well, you will not need to prepare many gels.
Teacher UPDATE (1/6/2009) for Lab 1: Problems with Warping Gel Trays
Many of our partners have mentioned a problem with gel trays “leaking” agarose. In most of these cases, the problem has been traced back to pouring the
agarose without first letting it cool. If the agarose is poured immediately after the agarose has melted, it is far too hot and will warp the gel tray. In some
cases, we have observed hot agarose actually causing the bonds between the bottom of the tray and its sides to dissolve.
The solution to this problem is to allow the agarose to cool to 60o C or below before dispensing into the gel tray. If the flask containing the melted agarose is
too hot to hold in your bare hands, it is too hot to pour into the gel tray.
Missing washers, from the screw holding the gates to the Fotodyne tray, can also cause the trays to leak agarose. Without the washer, the nylon screw can
“bottom out” and will not allow the gate to form a tight seal against the tray.
If the tray is too warped or you are missing one or more of these washers, you can use masking tape to seal the ends of the tray. Be certain to make your
site coordinator aware of these or any other equipment problems when you return the equipment to the distribution center.
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