Special Project Lab - Cloning
Genes using PCR
Goal:
Clone HOM-C genes from other nematodes, especially orthologs of the gene lin-39.
Background:
To be discussed in lab –
more written material to follow
[PCR is reviewed below.]
A List of tasks is shown
below:
1.
Design primers for PCR, based on alignments of known amino acid sequences.
2.
Isolate genomic DNA from organism.
3.
Amplify fragments of gene-of-interest using PCR. Analyze PCR results by gel
electrophoresis.
4.
Clone PCR fragments into vector (using TOPO-TA cloning).
5.
Transform, then respread traansformants, grow up overnight cultures.
6.
Purify plasmid DNA from candidate clones.
7.
Analyze clones initially by restriction digests.
8.
Select and prepare DNA from candidate clones for DNA sequencing.
9.
Send out DNA for sequencing, await results.
10.
Analyze sequence returned by BLAST, etc. Identify which HOM-C genes were
isolated.
Note that we may not be
able to perform everything listed in the limited time available.
A detailed flowchart for
a project like this is found on the next page, including alternate decisions to
be made based on results obtained along the way.
Note that the scheme
illustrated is somewhat general; some of the choices shown may not be relevant
to the exact project in a given year.
Polymerase Chain Reaction with Degenerate
Oligonucleotides
Lab Objectives
1) Learn about the theory and practice of the
polymerase chain reaction (PCR) using degenerate oligonucleotide primers to
isolate homologous genes from organisms where they have not yet been
identified.
2)
Extract genomic DNA for use in PCR.
3)
Set up PCR with a variety of conditions.
Polymerase Chain Reaction
This
extremely versatile and widely-used technique has been referred to as "the
Swiss army knife" of molecular biology. The technique was invented by Kary
Mullis, who was awarded the Nobel Prize for the technique, and is patented by
the biotechnology company Perkin-Elmer Cetus. One important use and perhaps its
greatest power lies in the detection of specific DNA sequences found at
extremely low concentration. Theoretically, PCR can be used to detect even a
single molecule of DNA. Hence, it has great utility not only in molecular
biology, but also in forensics*, paleobiology , and other fields. This power depends on the exponential amplification of
specific DNA molecules by repeated cycles of synthesis. (*Interestingly, Mullis
was called to testify at the O. J. Simpson trial, although he never actually
took the stand. DNA over 10
million years old has been amplified using PCR. The technique also provided the
scientific underpinnings of the science fiction novel and movie Jurassic Park, in which dinosaur DNA is amplified, sequenced
and reconstructed to resurrect these extinct reptiles.)
To understand how PCR
works, we must first review the basics of DNA structure and synthesis. DNA is a
double-stranded molecule in which complementary nucleotides pair between two
strands running in opposite directions (Fig. 1). An exact copy of a DNA
molecule can be made
5' -
TCTCAAACGTTAACGTAGCTAGCTCGGGCTAGCTCGCTAGATAGCT - 3'
||||||||||||||||||||||||||||||||||||||||||||||
3' -
AGAGTTTGCAATTGCATCGATCGATCCCGATCGTGCGATCTATCGA - 5'
Fig. 1. A double-stranded
DNA molecule. The numbers 3' and 5'
(pronounced "three prime" and "five prime") indicate
the direction of the DNA strand; the lines between the two strands indicate
complementary base-pairing.
by taking either single
strand and matching its sequence of bases with the complementary nucleotides.
This is normally accomplished in a cell (or molecular biologist's test tube) by
the enzyme DNA polymerase.
The enzyme requires three ingredients: template DNA (the DNA being copied), deoxynucleotide
triphosphates (DNA precursors:
dATP, dCTP, dGTP and dTTP), and a primer (a short length of single stranded DNA, or oligonucleotide, that complements a stretch of template DNA).
DNA polymerase can't make new DNA from a bare single strand of template - it
has to have some place to start. It synthesizes new DNA by adding nucleotides
to the end of the primer (Fig. 2).
Primer anneals to
complementary site:
5'- AACGTTAACGTAGCT - 3'
|||||||||||||||
3' - AGAGTTTGCAATTGCATCGATCGATCCCGATCGTGCGATCTATCGA
- 5'
New bases added by DNA
polymerase:
5'-
AACGTTAACGTAGCTAGCTCGGGCTAGCTCGCTAGATAGCT - 3'
|||||||||||||||::::::::::::::::::::::::::
3' -
AGAGTTTGCAATTGCATCGATCGATCCCGATCGTGCGATCTATCGA - 5'
Fig. 2 - A single-stranded
DNA molecule with a short double-stranded region. DNA polymerase can now add
new complementary nucleotides to the oligonucleotide primer starting at the 3'
end of the double-stranded region using deoxynucleotide precursors. (The ':'
simply indicates new base-pairs.)
Any PCR reaction has the
same basic sequence of events which are repeated:
Basic
Cycle Sequence:
Denaturation
Primer annealing
DNA synthesis
Repeat
Denaturation: Double-stranded template DNA must be denatured or "melted" into two separate single
strands before synthesis can occur. Since the base-pairing of DNA is very
stable, this must be done at a high temperature, near the boiling point of
water, for example 93ˇ C.
Primer annealing: After the DNA is denatured, primers must be
allowed to base-pair with the single-stranded templates to form those short
stretches of double-stranded DNA need for synthesis. This is done at a much
lower temperature (e.g., 40-70ˇ C), specific to the primers being used.
DNA synthesis: The temperature of the reaction is raised to
the optimal temperature for the DNA polymerase enzyme to synthesize new DNA.
The enzyme now adds deoxynucleotides to the 3' end of the primers, using bases
complementary to those in the template DNA. For the enzyme typically used for
PCR, Taq DNA
polymerase, this optimal
temperature is 72ˇ C.
Repeat: This is the key to the amplification of the DNA. This sequence of
events is repeated from 20-40 times to synthesize more and more DNA. This also
explains why we use a specific type of DNA polymerase for PCR. The high
temperature of the denaturation step will destroy DNA polymerase from most
animals or ordinary bacteria; therefore, we use a DNA polymerase isolated from thermophilic (Greek: heat-loving) bacteria. These bacteria
normally live at very high temperatures in hot springs or underwater volcanic
vents, some near the boiling point of water. The most commonly used DNA
polymerase for PCR, Taq polymerase, is from Thermus aquaticus.
The repeated cycles of
denaturation, annealing, and synthesis result in exponential amplification of
specific segments of DNA, given the proper conditions are fulfilled: two
primers anneal near to one another on opposite strands of a double-stranded
template DNA. Otherwise, only a linear amplification of the DNA can take place.
Follow the example illustrated below (Figure 3).
Fig. 3 - The first cycle of denaturation,
annealing and synthesis, starting with the double-stranded template shown
below.
5'-..TCTCAAACGTTAACGTAGCTAGCTCGGGCTAGCTCGCTAGATAGCTGATCCTCTCTCTGCATCCGT..-
3'
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
3'-..AGAGTTTGCAATTGCATCGATCGATCCCGATCGTGCGATCTATCGACTAGGAGAGAGACGTAGGCA..-
5'
Denature:
5'-..TCTCAAACGTTAACGTAGCTAGCTCGGGCTAGCTCGCTAGATAGCTGATCCTCTCTCTGCATCCGT..-
3'
3'-..AGAGTTTGCAATTGCATCGATCGATCCCGATCGTGCGATCTATCGACTAGGAGAGAGACGTAGGCA..-
5'
Anneal primers:
5'-..TCTCAAACGTTAACGTAGCTAGCTCGGGCTAGCTCGCTAGATAGCTGATCCTCTCTCTGCATCCGT..-
3'
|||||||||||||||
3'-
TAGGAGAGAGACGTA - 5'
5'-
AACGTTAACGTAGCT - 3'
|||||||||||||||
3'-..AGAGTTTGCAATTGCATCGATCGATCCCGATCGTGCGATCTATCGACTCGGAGAGAGACGTAGGCA..-
5'
Synthesize (new
base-pairings indicated with a ":"):
5'-..TCTCAAACGTTAACGTAGCTAGCTCGGGCTAGCTCGCTAGATAGCTGATCCTCTCTCTGCATCCGT..-
3'
::::::::::::::::::::::::::::::::::::::::::::::|||||||||||||||
<- GAGTTTGCAATTGCATCGATCGATCCCGATCGTGCGATCTATCGACTAGGAGAGAGACGTA - 5'
5'-
AACGTTAACGTAGCTAGCTCGGGCTAGCTCGCTAGATAGCTGATCCTCTCTCTGCATCCGT ->
||||||||||||||:::::::::::::::::::::::::::::::::::::::::::::::
3'-..AGAGTTTGCAATTGCATCGATCGATCCCGATCGTGCGATCTATCGACTCGGAGAGAGACGTAGGCA..-
5'
The key to PCR
amplification is that synthesis of the new strand from one primer makes a new
copy of the template for the other primer. (These are underlined in Figure 4.) Thus when the cycle is
repeated, note that the denaturation produces 4 single-stranded templates from
the original two:
Fig. 4 - Molecules at the end of the first cycle. Newly synthesized
primer annealing sites are underlined.
5'-..TCTCAAACGTTAACGTAGCTAGCTCGGGCTAGCTCGCTAGATAGCTGATCCTCTCTCTGCATCCGT..-
3'
::::::::::::::::::::::::::::::::::::::::::::::|||||||||||||||
<- GAGTTTGCAATTGCATCGATCGATCCCGATCGTGCGATCTATCGACTAGGAGAGAGACGTA - 5'
5'-
AACGTTAACGTAGCTAGCTCGGGCTAGCTCGCTAGATAGCTGATCCTCTCTCTGCATCCGT
->
||||||||||||||:::::::::::::::::::::::::::::::::::::::::::::::
3'-..AGAGTTTGCAATTGCATCGATCGATCCCGATCGTGCGATCTATCGACTCGGAGAGAGACGTAGGCA..-
5'
Next cycle: denature,
anneal and synthesize. Each new strand is italicized. All primer sites available
for the next round of synthesis are underlined (there are now eight.) Note also
that two of the synthesized strands are now of a defined length - the number of
bases from the 5' end of one primer to the 5' end of the other primer. In this
case, the length is 57 bases. This will be the length of essentially all the
amplified DNA molecules after several cycles.
5'-..TCTCAAACGTTAACGTAGCTAGCTCGGGCTAGCTCGCTAGATAGCTGATCCTCTCTCTGCATCCGT..-
3'
:::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::
<- GAGTTTGCAATTGCATCGATCGATCCCGATCGTGCGATCTATCGACTAGGAGAGAGACGTA - 5'
AACGTTAACGTAGCTAGCTCGGGCTAGCTCGCTAGATAGCTGATCCTCTCTCTGCAT - 3'
:::::::::::::::::::::::::::::::::::::::::::::::::::::::::
3'-..GAGTTTGCAATTGCATCGATCGATCCCGATCGTGCGATCTATCGACTCGGAGAGAGACGTA - 5'
5'-
AACGTTAACGTAGCTAGCTCGGGCTAGCTCGCTAGATAGCTGATCCTCTCTCTGCATCCGT..-
3'
:::::::::::::::::::::::::::::::::::::::::::::::::::::::::
3'- TTGCAATTGCATCGATCGATCCCGATCGTGCGATCTATCGACTAGGAGAGAGACGTA
5'- AACGTTAACGTAGCTAGCTCGGGCTAGCTCGCTAGATAGCTGATCCTCTCTCTGCATCCG ->
:::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::
3'-..AGAGTTTGCAATTGCATCGATCGATCCCGATCGTGCGATCTATCGACTCGGAGAGAGACGTAGGCA..-
5'
Note there are now 4
double-stranded copies of the original DNA template molecule (and 8 templates
for the next cycle - primer sites are underlined). Thus, the number of
molecules of the synthesized using the specific primers doubles with each
cycle. For a single starting molecule, the number of molecules at the end will
be 2n, where n is the number of
cycles. For a typical PCR reaction
with 40 cycles, that would be 240 =
1.2 x 1024 molecules. If only a
single primer is used, the number of molecules will increase linearly. That is,
starting with a single molecule, the number of molecules at the end would be
2n, or for 40 cycles, 2x40 = 80 molecules. This is represented in a drawing
below (Fig. 5)
Fig. 5 - Graphic representation
of DNA amplification by polymerase chain reaction. Number of copies of template
DNA double with each round (cycle) of synthesis.
Degenerate primer PCR
PCR
can also be used to amplify segments of DNA for which one does not know the
exact DNA sequence. For example, a scientist may know the amino acid sequence
of a bit of protein, but she would like to isolate the gene encoding that
protein. Or, to try to identify a homologous gene from a different organism,
the molecular biologist may 'guess' at the correct amino acid sequence based on
that known from another organism. Since the genetic code is
"degenerate," that is, more than one triplet codon may encode the
same amino acid, the amino acid sequence does not unambiguously identify the
DNA sequence. To perform PCR in
such a situation, the scientist usually employs degenerate oligonucleotide
primers; that is, a mixture of
primers with slightly different sequences, hoping that at least one of the
primers will anneal to the target DNA and prime the DNA polymerase reaction.
For
example, it has been proposed that homeodomain-containing genes may be
controlling anterior-posterior pattern formation in essentially all
multicellular organisms. By determining the conserved regions of amino acid
sequence in the proteins encoded by these genes, we can identify sequences that
we would expect to find in other organisms as well. For example, if we compare
the sequence of the 60 amino acid DNA-binding region of the Drosophila homeodomain proteins Antennapedia (Antp), Deformed (Def) and Abdominal-B (Abd-B), there are two regions in which several amino
acids in a row are identical in all three proteins (see Fig. 6 below). This
suggests these amino acids comprise important functional parts of the protein
and are thus likely to be the same in other organisms that use these proteins.
These amino acid sequences can be "reverse-translated" to yield all
possible DNA sequences that could encode the given amino acid sequence (Fig.
7). Primers of these "degenerate" sequences can then be synthesized
and used in PCR to try to detect genes encoding homeodomains in any organism
using whole DNA from that organism as template.
Fig. 6 - Comparisons of
amino acid sequences of three Drosophila homeodomain proteins. The top sequence of the Antp
homeodomain is shown in full (in single letter amino acid code).
Sequences below are indicated with a dash (-) when identical with the Antp amino acid. Different amino acids at that
position are indicated by the alternate amino acid letter. Two stretches of
amino acids that are identical in all three proteins are underlined in the Antp sequence.
Ant RKRGRQTTYTRYQTLELEKEFHFNRYLTFFFFIEIAHALCLTERQIKIWFQNRRMKWKKEN Def P--Q--A---H-I---------Y--------------T-V-S-----------------D- Abd VRKK-KP-SKF----------L--A-VSKQK-W-L-RN-Q-----V----------N--NS
If primers encoding part
of the sequences underlined in Fig. 6 are used, then DNA will be amplified to a
characteristic length of 3 x (total number of amino acids encoded by primers
and region in between). In the example above, 43 amino acids are included;
therefore the amplified fragment of DNA would be 129 base pairs in length. The
products of the PCR reaction can be run on an agarose electrophoresis gel to
separate DNA fragments by size and evaluated for the presence of DNA bands of
the predicted size. Primers 18-30 bases long are used typically for PCR
reactions.
Fig. 7 -
"Reverse-translation" of a conserved amino acid sequence from three Drosophila homeodomain proteins. (a) All possible codons
encoding the amino acid sequence KIWFQN. DNA sequences are indicated below the
amino acid sequence in lower case letters. (b) Sequence of degenerate
oligonucleotides encoding KIWFQN. At position 3 in the sequence, the base may
be either a or g; at position 6, the base may be either a, c
or t., etc. No matter what
the actual DNA sequence in the gene of interest, if the gene encodes KIWFQN, at
least one of the various oligonucleotides will hybridize and be able to prime
the DNA polymerase reaction. (c) Degeneracy of the oligonucleotide primer (the
number of different primers in the mix). To calculate degeneracy, multiply the
number of different bases at each position. Degeneracy of this primer is 48, an
acceptably low value. Degenerate PCR typically can work with degeneracy of each
primer at easily 1000-fold. To synthesize such a degenerate mix of primers is
easy, if you know how laboratory synthesis of single-stranded DNA occurs.
(a)
K I W F Q N
aaa ata tgg ttc caa aac
aag atc ttt cag aat
att
(b)
DNA base at position number:
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18
a a a a t a t g g t t c c a a a a c
g c t g t
t
(c) Degeneracy:
2 (base 3) x 3 (base 6) x 2 (base 12) x 2 (base 15) x 2 (base 18) = 48
Quick DNA Isolation for Polymerase Chain Reaction
To
perform a Polymerase Chain Reaction (PCR), you need very little template DNA
and it does not have to be especially pure or intact (this is why PCR is so
powerful in forensics and paleobiology). Nevertheless, PCR works better with
sufficient high quality DNA (clean and intact) than with little low quality DNA
(i.e., contaminated with proteins and significantly degraded). We will attempt
to purify DNA of sufficient quality and quantity to use as template in PCR.
Washing
worms and lysis procedure
1) Get a 60 mm plate of worms.
2) Wash the worms off the plate
with about 1.5 - 2 ml of cold water (nanopure H2O) and transfer to a labeled 1.5 ml centrifuge
tube. (One pasteur pipet-full is about 1.5 ml.)
3) Balance your tube with a partner
and centrifuge for 30 sec in a microcentrifuge at 2000-3000 RPM (~2000 g). Be
sure to balance the tubes by putting equal volumes of liquid in each tube and
putting them in opposite slots in the centrifuge rotor. An unbalanced
centrifuge will vibrate, rattle, or try to walk across the table. If that
happens, stop the centrifuge immediately and check the balance again.
4) After spinning, carefully remove
and discard the supernatant with a pasteur pipet (make sure not to suck up the
worms in the pellet) and re-wash the pelleted worms with 1.5 ml of water. Note: Worms don't make a hard pellet. DO NOT
disturb the pellet when removing
the supernatant or you will lose worms. Keep tip of the pipet away from the
bottom and try to remove all the supernatant in one action, without pipetting
fluid back into the tube. If you do resuspend the worm pellet, simply stop and
spin down again. Between spins, keep the tubes on ice. This will keep the worms
from wiggling around and breaking up the pellet.
5) Repeat the centrifugation and
washing procedure a total of three times. This procedure will remove most
bacteria that washed off the culture plates with the worms. See the next step
for special instructions for the final spin.
6) At the last centrifugation, spin
at maximum speed in microcentrifuge. Then remove as much fluid as possible
without removing the worms. You may want to use a P200 pipetman for the last
bit.
7) Add 220 µl cell lysis solution
(CLS) to which Proteinase K has been added (final concentration 1 mg/ml).
8) Mix with vortexer, place tube in
65ˇ C water bath.
9) Incubate for 40 - 60 minutes,
vortexing 2-3 times during the digestion. (If you have a lot of debris
remaining after the digestion, centrifuge 1 min at maximum speed in
microcentrifuge and transfer the supernatant to a clean tube using a pipettor
and pipet tip.)
Note that during long incubations,
you can be setting up your PCR tubes (see below), adding primers and water. Do
not add reaction mix until after your template is prepared and added to the
tubes.
10) Place on ice for 1 min, then
remove to room temperature.
RNAse treatment and Protein
precipitation
1) Add 1 µl RNAse A solution, mix
by gently inverting the tube several times.
2) Incubate at 37ˇ C for 15 min.
This step digests RNA, which can interfere with the PCR.
3) Add 75 µl Protein Precipitation
solution (PPS - Ammonium acetate), vortex for 15 sec to mix.
4) Place tube on ice for 5 min.
5) Centrifuge 3 min at maximum
speed in microcentrifuge. You should see a large white pellet after this
centrifugation. If you do not, then the tube is likely heating up too much
during the spin. Cool the tube again on ice, and if necessary spin in a cooled
microcentrifuge. Remove the tube immediately after the spin.
6) Gently pipet the supernatant to
a clean 1.5 ml tube; be sure to leave pellet of crud behind. You don't need all
the supernatant -- it is better to leave some behind than bring along
contaminating pellet. Be sure to use a plastic pipet tip; never transfer
with a glass pipet. Under high salt conditions, DNA adheres tightly to glass.
DNA precipitation, drying and
rehydration
1) Add 220 µl Isopropanol, mix by
inverting gently several times.
2) Centrifuge 1 min at maximum
speed in microcentrifuge; a small white DNA pellet is usually visible in bottom
of tube. Don't be overly concerned if you don't see a pellet. With smaller
yields of DNA there may be no obvious pellet. In fact, having a very large
white pellet can be a bad sign, indicating carry-over of impurities.
3) Carefully pipet (with a
pipettor) or pour out the supernatant; invert and drain tube on a Kimwipe.
After this and subsequent centrifugations, move quickly to remove the
supernatant. The longer you wait, the more likely the pellet is to come loose.
4) Add 200 µl 70% Ethanol, invert
several times to wash pellet.
5) Again, centrifuge 1 min at
maximum speed.
6) VERY carefully remove the
ethanol. Beware -- the pellet may be loose, so pour or pipet slowly and watch
the pellet at all times.
7) Invert and drain tube on a
Kimwipe, allow to air dry for 10-15 min. (You may put open in 37ˇ incubator in
a microtube rack to aid drying.) All ethanol must be evaporated before DNA
is rehydrated. You can sniff at the tube for traces of EtOH. Look carefully
for droplets.
8) Add 50 µl DNA hydration solution
("DHS"), incubate incubate 65ˇ C for 15 min. Flick the tube gently
with your finger several times to help resuspend the DNA.
9) Keep DNA tube on ice prior to
using in setting up PCR reactions.
Your template DNA should now be ready to use in
PCR. (In many cases, this solution will contain a high concentration of
substances that inhibit PCR. Therefore, the DNA solution should diluted twice
to make a 1:10 and 1:100 dilution.)
10) Label two tubes 1:10 and 1:100. Add 9 µl DNA
hydration solution to each tube, then add 1 µl DNA to the 1:10 tube. Mix and
remove 1 µl, add to the 1:100 tube and again mix.
Setting
up PCR
For each 20 µl reaction,
add to a 500 µl thin-walled microcentrifuge tube (see below):
1 µl primer 1
1 µl primer 2
1 µl template DNA
17
µl PCR reaction mix, containing
-
PCR reaction buffer concentrate diluted with H20
-
Deoxynucleotides ("dNTPs" - dATP, dCTP, dGTP and dTTP)
- Taq DNA Polymerase (1 Unit per reaction)
For each experimental
reaction, do the following controls:
Positive
control (+C):
C.
elegans DNA + primers 1 and 2
This tests control for the success of the PCR
reaction itself. Success using DNA that has been previously shown to work in
this reaction with these primers indicates a "real" failure if
nothing is seen in the experimental reaction. Add the +C DNA as the very last
item to help avoid contamination.
Negative
controls:
a) Template DNA + primer 1 alone (add 1 µl H20 more)
b) Template DNA + primer 2 alone
c) No template DNA + primers 1 and 2
Any DNA fragments detected in experimental
reactions that are also found in negative control reactions should be
discounted. Controls 2 and 3 test for spurious bands that can arise from only a
single primer. Control 4 tests for contamination of the reaction with an
amplifiable template DNA. This is a non-trivial problem with PCR because of its
great power to amplify very small amounts of DNA.
Note that ideally we would include single primer
controls like tubes 5 & 6 for each of the experimental template dilutions
(1X, 1:10, 1:100); we compromise here on the number of reactions needed by
doing these controls only on the 1:10 dilution of template.
To keep track of what must be added to each tube, make a grid like
the following:
Tube No.
|
Reagent |
1 |
2 |
3 |
4 |
5 |
6 |
7 |
|
Primer 1 |
1 µl |
1 µl |
1 µl |
1 µl |
none |
1 µl |
1 µl |
|
Primer 2 |
1 µl |
1 µl |
1 µl |
1 µl |
1 µl |
none |
1 µl |
|
water |
1 µl |
none |
none |
none |
none |
1 µl |
1 µl |
|
Template DNA |
1 µl (+C) |
1 µl (1X, Exptl) |
1 µl (1:10) |
1 µl (1:100) |
1 µl (1:10,
Exptl) |
1 µl (1:10,
Exptl) |
none |
|
Reaction mix |
17 µl |
17 µl |
17 µl |
17 µl |
17 µl |
17 µl |
17 µl |
Be sure to label your
tubes with a distinguishing label, such as your initials, in addition to
numbers.
In some cases the two
different primers may be already mixed – add 2 µl of the combined primers
(except for the single primer controls).
Using a Finnpipete
0.5µl - 10 µl pipettor:
1) Add 1 µl primer 1 to
each appropriate tube (you may use the same pipet tip),
close
lid, advance each tube to next row in rack.
2) Add 1 µl primer 2 to each appropriate tube
(use new pipet tip for each tube), advance tubes.
3)
Add 1 µl water to appropriate tubes (negative controls), advance tubes.
4)
Add 1 µl template DNA to each appropriate tube (new pipet tips), advance
tubes.
5)
Add 17 µl PCR reaction mix to each tube. As you add mix, close lid and put tube
on ice.
If using a thermocycler
without a heated lid, add step 6:
[6) Layer 30 µl mineral
oil on top of each reaction (Use Pipetman P-200).
Add
oil to the inside wall of each tube; it will flow down and cover the aqueous
reaction. Use a new pipet tip for each tube to avoid contamination. The oil is
necessary to keep the reaction mix from evaporating during the PCR cycling
(remember that the reaction will be repeatedly heated to near boiling). Return
tubes to ice.]
7) Bring tubes to PCR thermocycler. The
thermocycler will be pre-programmed for appropriate PCR protocol.
PCR
protocol:
94ˇ
C for 1 min
45-50ˇ
C for 1 min
72ˇ
C for 1 min
Repeat
40 cycles;
72ˇ
C for 5 min
Go
to 4ˇ C indefinitely (ŇrefrigeratesÓ tubes until they are removed.)
8) Open lid, and firmly press tubes into places
in PCR thermocycler. Activate appropriate thermocycler program (Instructor),
close lid, sigh with relief.
9) After reactions are complete, remove tubes and
store in freezer. Reactions will be analyzed by gel electrophoresis in the
following lab.
Gel
Analysis of PCR, Isolation of PCR bands
1. Thaw PCR tubes; keep on ice after
thawed.
2. Prepare an agarose gel (check for
the percentage), 30 ml 1X TBE gel with the 8 well comb.
3. Transfer 4 µl of each experimental
reaction (those using both primers and an ÔexperimentalŐ DNA template) to fresh
1.5 ml tubes for possible use later. (DO NOT add 6X GLB to these tubes.) Keep on ice.
4. Add 3.2 µl 6X GLB to the remainder (~16 µl) in each PCR tube.
5. Load 10 µl of each
reaction on gel. Load 10 µl "2-Log DNA ladder" as molecular weight
marker.
6. Run the gel at 150 V until the
first dye has reached ~2/3 length of gel.
7. Stain and photograph the gel.
Gel #2
Once
the above gel is running, prepare a second gel following instructions below.
This gel may be used to isolate a specific band from the PCR for subsequent
cloning. (The next steps are dependent on results of first gel. In interest of
time, we will prepare and begin running a gel that we may need after seeing the
first gel.) This gel should be prepared as cleanly as possible, and uses a
special buffer the components of which should not inhibit cloning reactions
used later.)
1. Wear gloves to set up this gel.
Wash all the gel apparatus components thoroughly with deionized water and wipe
with Kimwipes to remove any possible contaminants. Use the 8 well comb. One gel
will be sufficient for 2 students.
2. Make an agarose gel (same
percentage as above) with 30 ml of 1X modified TAE ("TA low E") -
this buffer has a ten-fold reduced EDTA concentration from traditional TAE,
which is frequently used in place of TBE buffer. EDTA can interfere with a
number of molecular biology reactions that require Mg++. Note
that the TAlowE buffer is NOT
reused.
3. Pool the remaining experimental
reactions (tubes 1 - 3) in a single tube. Load 10 µl "2-Log DNA
ladder" in an outermost lane, and load the reactions in an adjacent lane.
The second student do the same, on the opposite side of the gel (again, load
the MW marker in the outer lane, with the reactions loaded adjacent).
4. Use 1X TAlowE buffer for the gel.
Run at 150V.
5. If this gel is to be used, cut off
the outermost lane and a small portion of the lane containing your reactions. You may want to do this
under the direction of the instructor. Stain only this part of the gel with
EtBr. DO NOT stain the remainder of the gel.
6. Under UV light, with the unstained
portion of the gel blocked, line up the stained portion of the gel with the
unstained. Use a fresh scalpel blade to cut out the region of the unstained gel
corresponding to the PCR band you wish to clone. Ideally, the piece of gel cut
out should be no more than ~100 µl of gel (about 100 mg in mass). From this
point, proceed with the Millipore Ultrafree-DA protocol.
DNA
Extraction from Agarose Gels with the
UltrafreeŞ-
DA Centrifugal Filter Device
A P P L I C A T I O
N N O T E
The
Ultrafree-DA device is designed to recover 100 to 10,000 bp DNA from agarose
gel slices in one 10- minute spin. It consists of a pre-assembled sample filter
cup with agarose Gel Nebulizer, and a microcentrifuge vial. The device utilizes
gelcompression to extract DNA from theagarose. Centrifugal force collapsesthe
gel structure, drives the agarosethrough a small orifice in the Gel Nebulizer
and the resultant gel slurry is sprayed into the sample filter cup.
As
the agarose is compressed at 5,000xg, DNA is extruded from the gelÍs pores. The
gel matrix is retained by the microporous membrane, and the DNA passes freely
through the membrane. DNA can then be recovered in the filtrate vial. DNA prepared
with this device requires no further purification for most applications,
including cloning and radioisotopic or fluorescent DNA sequencing. Since
agarose gel elec- trophoresis has high resolving power, the small and large
non-specific amplification products that frequently interfere with cloning and
sequencing after PCR (polymerase chain reaction) are completely removed from
the product.
Materials
