Murray Lab

University of Iowa
Created: Unknown
Revised: February 2010, EL
Reviewed: May, 2010, EL; March, 2011

1
POLYMERASE CHAIN REACTION (PCR)

If you have never done PCR or have questions, please see:
Elizabeth Leslie (primary) or Aline Petrin (secondary)

PCR is used to amplify a segment of DNA that lies between two regions of known sequence.
This process involves three steps:
Denaturation: 94 for 30 sec. (allows double strands to separate)
Annealing: 55 for 30 sec. (allows primers to bind to DNA)
Extension: 72 for 30 sec. (the new DNA strand is synthesized extending from each
primer along the template)

PCR REAGENTS
Buffer:
Why do we use it? The buffer promotes Taq usage. If the pH of the reaction drops by
more than one full unit the reaction becomes more acidic and the enzyme fails to work.

dNTP's: Deoxynucleotide triphosphates
Why do we use it? These are incorporated into the newly synthesized DNA strands
during the extension step of PCR.

Taq Polymerase: From Thermus aquaticus a thermophilic, eubacterial microorganism.
Why do we use it? Taq is the enzyme involved in strand synthesis.
Characteristics:
- Sensitive to Magnesium.
- Capable of growth at 70-75 celsius.
- High temperature optimum for DNA synthesis.
- Rate of processivity is 35-100 nucleotides/second. Processivity= the number
of nucleotides synthesized before the enzyme dissociates from the template.

Oligonucleotide Primers: Sequences of DNA 18-24 bases long, which anneal to the template
strand of DNA allowing the Taq Polymerase to synthesize a strand complementary to the
template strand.

ALTERNATE REAGENTS
Alternative reagents may be used to increase PCR yield and specificity including DMSO,
alternate buffer or alternate polymerase enzymes.

Dimethlysulfoxide (DMSO) may be used in a PCR reaction to increase amplification. DMSO
helps loosen secondary structures that may be prohibiting the Taq polymerase from attaching
to the template strand. The standard volume used in a PCR reaction is 5% DMSO. For
example, in a 10 ul reaction volume, 0.5 ul DMSO is added. Consequently, 0.5 ul of water is
Murray Lab
University of Iowa
Created: Unknown
Revised: February 2010, EL
Reviewed: May, 2010, EL; March, 2011

2
removed so that the total volume is not changed. DMSO is useful when amplifying GC-rich
sequences due to their third H-bonds.
NOTE: DMSO is carcinogenic! Take special care to always have gloved hands when
using DMSO.

______________________________________________________________________

The primary brand of polymerase the lab currently purchases is Biolase. The Biolase kits come
with buffer, MgCl
2
and polymerase. They are stored at 80 prior to use, as are the stock dNTPs.
The lab also purchases enzymes that are designed for higher accuracy or longer product
synthesis. These enzymes are available upon request.




Murray Lab
University of Iowa
Created: Unknown
Revised: February 2010, EL
Reviewed: May, 2010, EL; March, 2011

3
2X Biolase Mix

Personal batches of 2X Biolase Mix should be made for PCR convenience. Prior to use, all
reagents should be thawed and vortexed well (except Taq) and spun down in a microfuge.
This brings any reagent down from the cap of the tube. It should be noted on PCR sheets
whenever new reagents are used for the first time.

Component For 10mL For 5 mL Final Concentration

ddH
2
O 7.14ml 3.57 ml
10X NH
4
Buffer 2.00ml 1.00 ml 2X
MgCl
2
600ul 300 ul 3 mM
dNTP(100mM stock) 40ul each 20 ul 400uM each
Taq @ 5U/ul 100ul 50 ul

Combine these in a conical tube and mix well. Aliquot 500ul of mix into labeled eppendorf
tubes for daily use. This prevents contamination, shortens freeze/thaw time and extends the
life of the mix.

























Murray Lab
University of Iowa
Created: Unknown
Revised: February 2010, EL
Reviewed: May, 2010, EL; March, 2011

4
OPTIMIZING PRIMERS (Gradient PCR)

Rehydrating primers and making dilutions:
1. When you receive your primers from IDT, they will be dry in the bottom of a blue-cap
tube. Spin the tubes to make sure the primer is down at the bottom.

2. Rehydrate primers with ddH
2
O. We want the primers to be 100 uM stocks, so add ten
times the amount of primer listed in nmol on the tube. For example, if your primer tube
says 32.0 nmol, add 320 ul of water. Vortex really well. Really well! Your primer must
be properly resuspended or dilutions will not be accurate.

3. Make a 20 uM dilution to use for PCR. Use the label from the primer info sheet for new
screw-top tubes. Add 20 ul of 100 uM stock and 80 ul ddH
2
0 to the tube. Vortex!

Optimizing primers:
Primers must be optimized before you can use them to with your samples to determine the
appropriate conditions for the PCR reaction. To do this we use the MJ thermocycler and run a
gradient PCR which varies the annealing temperature across each column of the plate. This
allows us to test the quality of the PCR reaction for a range of temperatures at the same time.








We test primers on CEPH DNA. Pick a sample from the old CEPH DNA boxes and dilute an
Murray Lab
University of Iowa
Created: Unknown
Revised: February 2010, EL
Reviewed: May, 2010, EL; March, 2011

5
aliquot to 20 ng/ul.

1. Sign up for the MJ by writing your name and drawing an arrow for the time slot for
which you want to reserve. A gradient PCR reaction will take about 3 hours.

2. Print out a Gradient PCR reaction sheet. (gradient PCR form.doc)

3. Label a master mix tube for each primer to be tested.

4. Remove reagents from freezer boxes and allow them to thaw on ice. Vortex everything
well once fully thawed. Keep biolase on ice.

5. Label plate with appropriate information. For example: primer name, date, DNA plate,
and your name.

6. Add 1 ul of DNA to each well of the plate you will be using. Do not use a repeat
pipettor, it does not dispense 1 ul accurately. Use the multichannel.

7. Make the Master Mix by combining the appropriate amounts of each reagent into the
Master Mix labeled tube. Mix the master mix well by pipeting up and down with the
p200 set at 200ul or by briefly vortexing (one pulse!). Check off each reagent as you add
it.

8. Distribute the master mix to the plate. This may be done with a motorized pipetman.
The first master mix tube will be for the first row (i.e. row A), the second tube is for the
second row (i.e. row B).

9. Seal the plate with the plastic PCR covers. Use the plate sealer around all of the edges.
You may seal with two covers if evaporation is a problem. Spin down the plate.

10. Place reactions in the MJ PCR machine.

11. You will need to edit the program to select the gradient you want to use. Use the
arrows to select Edit. Scroll to the program Grad. Use the arrows and enter to
scroll to the annealing temperature (where it says X to Y). Type in the temperatures
you want for the low and high ends. 52-62 is a good starting point. If your primers have
a high melting temperature (Tm listed on the tube label), you can go higher (i.e. 55-65).
There is no reason to go below 50 degrees or above 68 degrees. Keep scrolling through
the program to check it until you return to the main screen.

12. Now you are ready to run the PCR reaction. Select Run. Select Grad. It will ask if
you are running tubes or plates, select plate. Enter 10 ul for the volume. ALWAYS USE A
HEATED LID.
Murray Lab
University of Iowa
Created: Unknown
Revised: February 2010, EL
Reviewed: May, 2010, EL; March, 2011

6

13. After your program has finished running, the temperatures for each column will be
listed. Write them down!

14. If you need to figure out what the temperatures were after that, select List. Then
Gradient Calculator. Enter the low and high temperatures.





































Murray Lab
University of Iowa
Created: Unknown
Revised: February 2010, EL
Reviewed: May, 2010, EL; March, 2011

7
INITIAL PCR

1. Sign up for a PCR machine by writing your name and drawing an arrow for the time slot
for which you want to reserve. A standard 45 sec/45 sec/45 sec program will take about
2 hrs.
- You must initiate your PCR cycles within 30 minutes of the time you signed
up for or forfeit the slot to the person signed up after you.
- If running late, it is common courtesy to let the person signed up after you
know when you started the thermal cycler.

2. Print out your Initial PCR reaction sheet. (initial pcr form.doc)
- Fill in the required information: Date, DNA Plate used, Gene, Exon/region,
primer names, annealing temperature, extension time, number of cycles, and
PCR machine used. Also include if new reagents are used. This information
is critical to track possible machine breakdowns, contamination of DNA
plates, or faulty reagents (ex. operator error in making biolase)
- Info on these sheets contains a list of all ingredients in a pcr reaction.

3. Calculate the volume of each reagent for the Master Mix if using less than a full plate of
DNA, otherwise follow the 100x calculations on the reaction sheet.
- A Master Mix is used to increase the volume needed of each reagent in order
to make pipeting easier and increase its accuracy.
- A Master Mix is the volume of each reagent multiplied by the number of
samples to be pcr'd plus a few extra to allow for loss of volume due to
pipeting.

4. Remove reagents from freezer boxes and allow them to thaw on ice. Vortex primers
well once fully thawed. Keep biolase on ice.

5. Label plate with appropriate information. For example: primer name, date, DNA plate,
and your name.

6. Aliquot the DNA into the plates. When pipetting from a stock plate always use a
multichannel pipette and check the tips to make sure you have pulled up DNA and
emptied the tip into the well.

7. Make the Master Mix by combining the appropriate amounts of each reagent into the
Master Mix labeled tube. Mix the master mix well by pipeting up and down with the
p200 set at 200ul or by briefly vortexing (one pulse!). Check off each reagent as you add
it.

8. Distribute the master mix to the plate. This may be done with a motorized pipetman
(repeater).
Murray Lab
University of Iowa
Created: Unknown
Revised: February 2010, EL
Reviewed: May, 2010, EL; March, 2011

8

9. Seal the plate with the plastic PCR covers. Use the plate sealer around all of the edges.
You may seal with two covers if evaporation is a problem. Spin down the plate.

10. Place reactions in the pcr machine and run the appropriate program for the primers
used. (Always double check the pcr program on the machine to ensure that it hasnt
been changed.) You may want to wait until the machine has reached 94C before you
put the rxn in. This is called hot-start and prevents the rxn from occurring at temps
other than the specific ones you want.

a. If there is a finished PCR reaction in the machine, take the plate out and put it in
the blue yesterdays PCR box in the freezer.