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University of Iowa
Murray Lab
University of Iowa
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, MgCl2 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.
Created: Unknown
Revised: February 2010, EL
Reviewed: May, 2010, EL; March, 2011
Murray Lab
University of Iowa
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
ddH2O
10X NH4Buffer
MgCl2
dNTP(100mM stock)
Taq @ 5U/ul
7.14ml
2.00ml
600ul
40ul each
100ul
3.57 ml
1.00 ml
300 ul
20 ul
50 ul
2X
3 mM
400uM each
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.
Created: Unknown
Revised: February 2010, EL
Reviewed: May, 2010, EL; March, 2011
Murray Lab
University of Iowa
We test primers on CEPH DNA. Pick a sample from the old CEPH DNA boxes and dilute an
Created: Unknown
Revised: February 2010, EL
Reviewed: May, 2010, EL; March, 2011
Murray Lab
University of Iowa
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.
Created: Unknown
Revised: February 2010, EL
Reviewed: May, 2010, EL; March, 2011
Murray Lab
University of Iowa
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.
Created: Unknown
Revised: February 2010, EL
Reviewed: May, 2010, EL; March, 2011
Murray Lab
University of Iowa
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).
Created: Unknown
Revised: February 2010, EL
Reviewed: May, 2010, EL; March, 2011
Murray Lab
University of Iowa
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.
Created: Unknown
Revised: February 2010, EL
Reviewed: May, 2010, EL; March, 2011