Chem41 Lab
Week 2
Spring 2003
Outline:
1. Get the PCR digestion running
2. Pour the agarose gel
3. Run the PCR digest and the pET28a digest on the gel
4. Weigh out LB media ingredients
5. Make up kanamycin stock solution
6. Isolate the gel purified DNA fragments from the gel
1. Digestion Protocol for PCR product
PCR Product
Order addition
10 µl DNA
2 µl 10X NEBuffer 2
1 µl Nde1
1 µl Hind III
6 µl H2O
3
2
4
5
1
20 µl TOTAL
Incubate the Reaction mixture for 1hr at 37 C.
Enzyme reagents supplied with 10X NEBuffer 2. Working conc. 1X
NEBuffer 2.
Enzyme Stock Con. 20 U/µl (20,000 U/ml)
Working Con. 1 U/µl (1000 U/ml)
2. DNA gel protocol:
DNA gels are run in the presence of ethidium bromide, and nice little toxic molecule that
intercalates in between DNA base pairs and the causes DNA to glow when viewed with UV
light. This is great as it allows us to see the DNA, but ethidium bromide is not picky…it
would just as soon intercalate into YOUR DNA and cause you to glow. Of course, this
might also cause nasty mutations when your cells divide. So please wear gloves and
handle DNA gels and gel buffers with care!
While they are incubating, we will pour a DNA gel. A DNA gel, or agarose gel, is a mixture of a
buffer (TAE – Tris-acetate with EDTA) and agarose (a polysaccharide extracted from seaweed).
We’ll use 1% agarose and make 50 ml. The recipe for TAE buffer is below, but we’ll make this
one for you. The TAE-agarose must be heated up to near 100C in order for the agarose to
dissolve. Take care handling hot things! Once the gel solution cools to below 60C (otherwise it
will melt the gel box!), ethidium bromide is added and it can be poured into the gel mold. Combs
are inserted to make wells in which the DNA digest will be placed. The solidified gel is then
placed into the gel box, covered with TAE contaiing ethidium bromide, the digested plasmid DNA
is loaded (in a blue colored loading buffer), and an electric current is run through it. Since DNA is
NEGATIVELY charged, it will move toward the POSITIVE terminal, which is RED. If you run the
gel the wrong way, your DNA will run into the buffer, never to be seen again. The gels will run
about an hour. As we only have two power supplies, we’ll need three or four gels to a supply.
Instructions for the gel box are attached. In short, we’ll load a DNA ladder so that we can see how
big the bands are on the band and run for about 45-60 minutes at 150 V. Then we will use a
scalpel to cut the correct bands out of the gel, weigh them, and freeze them for use next week.
We can visualize the DNA by looking at the gel with a UV light box. The ethidium bromide causes
the DNA to glow visibly. Remember to take a picture for your lab notebook.
UV light will damage your eyes if you look at it unshielded. Make sure you wear protective
eyeware!
Setting Up the Gel:
1. Remove the SuperSafeTM lid from the gel box by pulling the lid upward. The cover is attached
to the unit at the connection of the power cords to the banana plugs. Level unit so that the gel
cast will be of even thickness.
2. Gel casting:
For models F8-58-710:
For shipping and convenient storage, the gel tray is packaged inside the unit. To remove the gel
tray, hold the unit firmly with one hand; grasp the long sides of the UVT gel tray and pull up slowly
at an angle. The tray needs to fit snug for leakproof gel casting, so it may be somewhat tight.
"Walking" the tray upwards at an angle may be helpful.
To cast gels, place the gel tray into the chamber so that the gasketed ends press against the
walls of the buffer chamber. Make sure the gel tray is pressed all the way down and rests level on
the unit's platform.
3. Preparing the gel:
The % of agarose and the buffer used is determined by the size of the samples to be separated
and further recovery of the samples (See Tables 2, 3 and 4). The agarose and buffer are mixed
and heated over a heat source, in a microwave oven, or in an autoclave until the agarose is
completely dissolved. The prepared gel then must be cooled to below 60.C before casting to
avoid warping the UVT gel tray due to excessive heat. If numerous gels are to be run in one day,
a large volume of gel may be prepared and be placed in a covered bottle stored between 40-60.C
in a water bath. This provides a ready gel supply in a warm liquid form that will solidify quickly
when gels are cast.
4. Pour or pipet the correct amount (See Table 3) of warm agarose (>60.C) onto the UVT gel tray
that has been placed into position in the gel box. Immediately after pouring, insert the desired
comb or combs into the comb slots to form the sample wells. To make wider lanes, use tape to
cover the gap between the narrower lanes of the comb. Allow the gel to solidify completely. A
single comb may be placed in either groove on the gel tray. If only a small portion of gel is
required for proper sample separation, then 2 combs may be used. This also expands the
number of samples per gel that may be run. To conserve agarose, a wall comb may also be used
to divide the gel and cast gels of varying length. Standard agarose should solidify completely in
about 30 minutes. If low melting point or a specialty agarose is used, consult the instructions that
came with the product.
Operating
For the F8-58-710:
1. Once the gel is completely solidified, lift the tray out of the chamber, turn it 90 degrees, and
replace it in the chamber with the first comb closest to the cathode ir black side. This running
position exposes the open ends of the agarose to the buffer.
2. Pour running buffer into the unit to fill chamber and completely cover and submerge the gel. A
"Fill Line" is located on each unit to clearly mark the correct buffer level. (See Table 1:
Recommended Running Conditions) for approximate buffer volumes needed for your unit. Too
little buffer may cause the gel to dry out during the run, while excess buffer may slow DNA
migration in the gel.
3. Carefully remove the comb (or combs) by tapping lightly to loosen, and slowly lifting out of the
gel tray to avoid damage to the wells.
4. Load prepared samples into the wells. Samples should be mixed with a sample loading buffer;
giving weight to the samples so that they drop evenly into the wells, and contain tracking dye to
monitor the gel run. See available comb section for approximate well volumes.
5. Place the lid with attached power cords onto the unit. This will connect the power cords to the
banana plug electrodes and complete the circuit. Plug the other end of the power cords into
appropriate power supply.
6. Turn on the power supply (See Table 1: Recommended Running Conditions). Carefully monitor
the gel run to avoid samples running into the path of another set of samples.
Finishing Up
When the gel run is complete and tracking dye has migrated as far through the gel as desired or
to the end of the gel, turn off the power supply and slide off the lid to disconnect from the power
source. Carefully remove the tray containing the gel (wear gloves if ethidium bromide is present).
The UV transparent gel tray makes visualization and photography with a UV light source easy
without the need to remove the gel from the tray.
Care and Cleaning
The gel box and tray should be rinsed under warm running water after each use. Rinsing the gel
tray will avoid any salt buildup in the gasketed material from the electrophoretic running buffer,
extending the gasket life and ensuring leak-free gel casting. Warning: Do not use ethanol or other
organic solvents to clean acrylic products. Organic solvents cause acrylic to "craze" or crack.
Clean all FisherBiotech acrylic systems by rins- ing with warm water, or clean with warm water
and a mild detergent to get rid of any debris. Never autoclave, bake or microwave this system.
Selection of Reagents
1. Agarose:
There are various types of agarose commercially available that may be
used. Besides standard ultra-pure electrophoresis grade agarose, there are also numerous lowmelting-point products for easy sample recovery, as well as specialty products formulated for
specific uses, i.e. to separate and/or recover very small or very large fragments.
NOTE: An increased agarose % provides better separation of small fragments and bands very
close together that tend to be more difficult to separate.
Example: A good mid-range gel % would be 0.7%, or 0.7g agarose in 100mL of electrophoresis
buffer (TBE or TAE), following heating and dissolving the agarose, 10uL of ethidium bromide
stock solution (5mg/mL) is added. The gel would be run with compatible electrophoret- ic running
buffer (1 X TBE or 1 X TAE) that also contained ethidium bromide. One liter of the running buffer
would contain 100uL of this 5mg/mL ethidium bromide stock solution.
Gel volume is determined by the following formula and may be adjusted according to need or
preference: gel width (cm) x gel length (cm) x gel thickness (cm) = mL of agarose
Ethidium Bromide:
For photodocumentation of samples, the gel may be stained during or following the run with a
variety of stains. The most common stain for DNA is ethidium bromide. Ethidium bromide may be
added directly to the gel and running buffer to visualize and photograph the separated fragments
following the gel run without the need for an additional staining step. The ethidium bromide is
added to both the gel (after heating) and the electrophoresis buffer at a concentration of
O.5ug/mL. Conversely, the gel may be stained in a concentrated ethidium bromide solution after
the gel run and rinsed for visualization.
NOTE: Ethidium bromide is a potential carcinogen. Care in handling the powder and stock
solution must be taken. Always wear gloves when handling the powder, solutions, and all gels
that contain ethidium bromide.
Buffers:
TAE Buffer:
-Use when DNA is to be recovered
-For electrophoresis of large (>20kb) DNA
-Applications requiring high resolution
-Has low ionic strength and low buffering capacity- recirculation maybe necessary for long runs
(>4 hrs)
This buffer is used because it has a basic pH, which gives the phosphate group of the DNA a net
negative charge allowing migration of the DNA toward the positive anode in the electrophoresis
chamber.
TAE- Tris-Acetate with EDTA
(40mM Tris base, 40mM acetic acid, 1 mM EDTA)
50x stock solution, pH-8.5
242g Tris base
57.1 mL glacial acetic acid
18.61g Na2EDTA * 2H2O (MW 372.24)
Distilled H2O to 1 liter final volume
1x working solution
40mM Tris-Acetate
1 mM EDTA
DNA Markers
Markers are run on each gel to monitor sample separation and to provide an accurate size
estimation of the samples. By running a known marker of a specific concentration, the amount of
the DNA can be estimated. These size markers are a suitable restriction digest of commonly
available DNA.
4. Bacterical LB media:
We will need 1 a total of 1.3 liters. We will not be autoclaving this week, so today just
weigh out the ingredients into a large flask.
Liquid LB (makes 1 liter):
To 950 ml H2O, add:
10 g tryptone
5 g yeast extract
10 g NaCl
Shake until dissolved, then pH to ~7 by adding 200 μl 5N NaOH.
Adjust to 1 liter with H2O
Autoclave for 20 minutes at 15 psi.
Allow to cool to 50-60 C before adding antibiotics.
LB for plates (we’ll need 200 ml of the liquid LB above for this):
Assume 30-35 ml per 90 mm plate.
Before autoclaving, add 15g/l bacto agar to LB.
Autoclave, cool, and and antibiotics as above.
5. Antibiotics:
pET28a contains the kanamycin gene, so we’ll need a stock solution.
Stock solution is 10 mg/ml in H2O.
Working concentration is 25 ug/ml.
6. QIAquick Gel Extraction Kit
Protocol
using a microcentrifuge
This protocol is designed to extract and purify DNA of 70 bp to 10 kb from standard or
low-melt agarose gels in TAE or TBE buffer. Up to 400 mg agarose can be processed per spin
column. This kit can also be used for DNA cleanup from enzymatic reactions (see page 8).
For DNA cleanup from enzymatic reactions using this protocol, add 3 volumes of Buffer
QG and 1 volume of isopropanol to the reaction, mix, and proceed with step 6 of the
protocol. Alternatively, use the new MinElute Reaction Cleanup Kit.
Notes: • The yellow color of Buffer QG indicates a pH 7.5.
• Add ethanol (96–100%) to Buffer PE before use (see bottle label for volume).
• Isopropanol (100%) and a heating block or water bath at 50°C are required.
• All centrifugation steps are carried out at 13,000 rpm (~17,900 x g) in a
conventional table-top microcentrifuge.
• 3 M sodium acetate, pH 5.0, may be necessary.
1. Excise the DNA fragment from the agarose gel with a clean, sharp scalpel.
Minimize the size of the gel slice by removing extra agarose.
2. Weigh the gel slice in a colorless tube. Add 3 volumes of Buffer QG to 1 volume of
gel (100 mg ~ 100 µl).
For example, add 300 µl of Buffer QG to each 100 mg of gel. For >2% agarose
gels, add 6 volumes of Buffer QG. The maximum amount of gel slice per QIAquick
column is 400 mg; for gel slices >400 mg use more than one QIAquick column.
3. Incubate at 50°C for 10 min (or until the gel slice has completely dissolved). To help
dissolve gel, mix by vortexing the tube every 2–3 min during the incubation.
IMPORTANT: Solubilize agarose completely. For >2% gels, increase incubation time.
4. After the gel slice has dissolved completely, check that the color of the mixture is
yellow (similar to Buffer QG without dissolved agarose).
If the color of the mixture is orange or violet, add 10 µl of 3 M sodium acetate,
pH 5.0, and mix. The color of the mixture will turn to yellow.
The adsorption of DNA to the QIAquick membrane is efficient only at pH 7.5.
Buffer QG contains a pH indicator which is yellow at pH 7.5 and orange or violet at
higher pH, allowing easy determination of the optimal pH for DNA binding.
5. Add 1 gel volume of isopropanol to the sample and mix.
For example, if the agarose gel slice is 100 mg, add 100 µl isopropanol. This step
increases the yield of DNA fragments <500 bp and >4 kb. For DNA fragments
between 500 bp and 4 kb, addition of isopropanol has no effect on yield.
Do not centrifuge the sample at this stage.
6. Place a QIAquick spin column in a provided 2 ml collection tube.
7. To bind DNA, apply the sample to the QIAquick column, and centrifuge for 1 min.
The maximum volume of the column reservoir is 800 µl. For sample volumes of more
than 800 µl, simply load and spin again.
8. Discard flow-through and place QIAquick column back in the same collection tube.
Collection tubes are re-used to reduce plastic waste.
9. (Optional)
10. To wash, add 0.75 ml of Buffer PE to QIAquick column and centrifuge for 1 min.
Note: If the DNA will be used for salt sensitive applications, such as blunt-end ligation
and direct sequencing, let the column stand 2–5 min after addition of Buffer PE,
before centrifuging.
11. Discard the flow-through and centrifuge the QIAquick column for an additional 1 min
at 13,000 rpm (~17,900 x g).
IMPORTANT: Residual ethanol from Buffer PE will not be completely removed unless
the flow-through is discarded before this additional centrifugation.
12. Place QIAquick column into a clean 1.5 ml microcentrifuge tube.
13. To elute DNA, add 30 µl elution buffer to the center of the QIAquick membrane,
let the column stand for 1 min, and then centrifuge for 1 min.
IMPORTANT: Ensure that the elution buffer is dispensed directly onto the QIAquick
membrane for complete elution of bound DNA. The average eluate volume is 48 µl
from 50 µl elution buffer volume, and 28 µl from 30 µl.
Elution efficiency is dependent on pH. The maximum elution efficiency is achieved
between pH 7.0 and 8.5. When using water, make sure that the pH value is within
this range, and store DNA at –20°C as DNA may degrade in the absence of a
buffering agent. The purified DNA can also be eluted in TE (10 mM Tris·Cl, 1 mM
EDTA, pH 8.0), but the EDTA may inhibit subsequent enzymatic reactions.