Note to the Reader Short Course on Quantitative Neuroanatomy
Organizers: John H. Morrison and Patrick R. Hof
Society for Neuroscience Education Committee
Saturday, November 7, 1998 (2:30–3:00 pm)
Westin Bonaventure Hotel and Suites, Los Angeles, CA

Protocol Details
Neuroscience Meets Quantitative Genetics: Using Morphometric Data to Map Genes that Modulate CNS Architecture

Robert W. Williams, Lu Lu, and Richelle C. Strom
Center for Neuroscience, Department of Anatomy and Neurobiology, University of Tennessee, Memphis Tennessee 38163
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PCR protocol

The methods detailed below can be used to detect differences of 4 bp or more in the length of PCR reaction products between 90 and 160 bp.

We amplify 50 ng of genomic DNA per reaction. Amplifying more DNA than this may result in overloaded lanes. Each individual 10 µl PCR reaction mixture contains the following:


  1. 1 µl of genomic DNA at a concentration of 50 ng/µl
  2. 2 µl of microsatellite primer stock solution (20 µl of both front and back primers from Research Genetics Inc. are combined and diluted in 160 µl of distilled water to make our primer stock solution)
  3. 7 µl of the PCR mix listed below

The third item above, the PCR mix, is usually made in large quantity and frozen until use (1.4 ml of reaction mix in a 1.5 ml polypropelene microcentrifuge tube is enough to run 2 x 96 reaction products). Multiply the volumes listed below by 100 for each set of 96 reactions. PCR mix consists of:


  1. 1.0 µl of 10X PCR buffer
  2. 0.8 µl of 25 mM MgCl2
  3. 0.1 µl of 80 mM dNTPs (20 mM each)
  4. 1.83 µl of 60% Ficoll (Type 400DL; Sigma F1418) in 1 mM cresol red
  5. 3.22 µl of filtered (0.45 µm) distilled water
  6. 0.05 µl of Taq at a concentration of 5 units/µl

We have been using MJ Research polypropylene V-bottom 96-well microplates sealed with a PCR plate sealer film (Cycleseal Cat# 1044-39-4 Robbins Scientific, 814 San Aleso Ave Sunnyvale CA 94086, (408) 734-8500). In our hands this plate sealer works better than MJR sealer.

DNA is amplified using a touchdown PCR protocol with the following cycling parameters:


  1. 94 deg for 3 min (melts duplex genomic DNA)
  2. 60 deg for 1 min (highest stringency annealing of primers)
  3. 72 deg for 1 min (extension of Taq across the target microsatellite)
  4. 94 deg for 30 sec
  5. 58 deg for 1 min (-2 deg stringency)
  6. 72 deg for 1 min
  7. 94 deg for 30 sec
  8. 56 deg for 1 min (-4 deg stringency)
  9. 72 deg for 1 min
  10. 94 deg for 30 sec
  11. 54 deg for 1 min (-6 deg stringency)
  12. 72 deg for 1 min
  13. 94 deg for 30 sec
  14. 52 deg for 1 min (-8 deg stringency)
  15. 72 deg for 1 min
  16. 94 deg for 30 sec
  17. 50 deg for 1 min (-10 deg = baseline stringency)
  18. 72 deg for 1 min
  19. Repeat Steps 16 through 18, a total of 30 times for a total of 35 cycles.
  20. Hold product at 8 deg in the machine and at 4 deg in the refrigerator until electrophoresis. This thermal cycler program run takes just under 2 hours to run on MJR PTC-100 or PTC-200.


The gel is made of 2.5–3.0% MetaPhor agarose (FMC Corp, mixed in 1X TBE buffer. Ethidium bromide at a concentration of 0.5 µg/ml is added to fresh gel before polymerization (typically 300 ml of buffer; 9 gm of MetaPhor agarose, 15 µl of 10 mg/ml EtBr). We cast gels for use with the Bio-Rad Subcell Model 192 electrophoresis system (Bio-Rad, 1-800-424-6723). These gels are large and relatively thin (gel dimensions are 25.5-cm-tall, 24.5-cm-wide and 3- to 4-mm-thick). Gels are cast with a total of 204 wells arranged in four rows of 51 wells. Only 48 wells per row are typically used. Each well is small (0.75 mm front-to-back, 3 mm right-to-left, and 2.5 mm deep). Adjacent wells are separated by 4.5 mm—two times the distance between the tips of the 12-channel multipipetter.

All of the PCR reaction product (now usually about 8.5 µl) is loaded into a well using a 12-channel pipette (Eppendorf or equivalent) and 0.1–10 µl tips (Fisher Redi-Tip, Cat # 21-278-2). PCR products in the eight rows of the 96-well plate are interleaved across rows of the gel: Row A to odd lanes 1 to 23; row B to even lanes 2 to 24, row C to odd lanes 25 to 47, row D to even lanes 26 to 48, etc.

Gels are run at 170 volts—equal to about 6.7 V/cm. Under these conditions the PCR products are usually well separated after 80 minutes. Note that the voltage gradient can be raised as high as 16 volt/cm to shorten time and improve band resolution. The gels are loaded at a temperature of 15 degrees while immersed in 1X Tris/Boric acid/EDTA buffer. We circulate the gel buffers through a cooling bath (Fisher Isotemp refrigeration unit 1016S with a Bio-Rad variable speed fluid pump) to maintain gel temperature at 18 to 20 deg. After the run is complete, gels are moved onto a large UV illuminator and cut apart into managable-sized pieces that can be photographed using a simple Polaroid system.

For PCR we use a set of 4 PTC-100 and PTC-200 cyclers with hot-top 96-well blocks

Problems: If the intensity of the PCR product is too low to photograph well, then try these simple modifications: 1. Increase the percentage of ethidium bromide in the gel two--fold (see paragraph above). 2. Another tactic is to simply run the entire PCR cycle again (another 35 cycles) without addition of any reagents. A more mind alternive is just to add 10 to 14 cycles.

Economizing: MetaPhor agarose is expensive ($2 per gram). We typically recycle all parts of gels that do not contain abundant amounts of PCR product. Large slabs are remelted and used again and again. In each cycle of recycling we typically add 2 µl of a 10 mg/ml solution of EtBr.

Photography is also expensive. An entire gel with 192 lanes can be photographed using just two exposures. The gel is first cut vertically down the middle. Then the important parts are sliced out and arranged close to each other in four rows (11 cm wide and 15 cm high) on a UV transilluminator (Fisher FBTIV-614). We use an Eicker brain knife to slice gels. The gel left-overs are put in a 1000 ml beaker for eventual recycling. A small Fotodyne hood (Cat 5-5342) is used with a hand-held Polaroid instant camera. Exposures are 0.5 sec at F8. Film is Polaroid Polapan 667 (3.25 by 4.25 inch).

We have designed Excel and FileMaker files that allow rapid entry of genotype data using a numeric keypad (8 = high band homozygote, 2 = low band homozygote, 5 = heterozygote).

Trouble-shooting: If amplification of PCR product is not uniform or the apparent concentration of the product on the gel is too low, assess the following factors:

  1. Make sure that the concentration of DNAs used in the reaction is relatively uniform. This will involve diluting each sample individually (our target concentration is 50 to 100 ng/per microliter).
  2. Preparing the MetaPhor gel can be critical. Make sure that the MetaPhor is dissolved completely: no bubbles or blocks of half-melted Metaphor. First dissolve in cold buffer and warm slowly with a stir bar, and after the Metaphor has dissolved completely put into microwave oven (high power: 1 min, wait 30 sec: 1 min then wait 30 sec, 1 min + 30 sec, 30 sec, 30 sec, 30 sec, 10 sec, etc. Times depend on volume). Be careful that the Metaphor does not boil over.
  3. Make sure the DNAs used in the PCR reactions are completely mixed before you pipette them into the PCR mix. Vortex and also mechanically stir with the pipette tip.
  4. Try doubling the concentration of Taq in the reaction.

Our thanks to Drs. Xiyun Peng, Guomin Zhou, Jing Gu, and Lu Lu for improving the efficiency and reliability of these methods.


  1. Laird PW, Zijderbeld A, Linders K, Rudnicki MA, Jaenisch R, Berns A (1991) Simplified mammalian DNA isolation procedure. Nucleic Acid Res 19:4293.
  2. White HW, Kurukawa N (1997) Agarose-based system for separation of short tandem repeat loci. BioTechniques 22:976–979.
  3. A great web site for trouble-shooting PCR reactions


Figure 2
Fig. 2.   Genotypes at a microsatellite locus on chromosome 5 (D5Mit294). The left lane is a DNA size standard, and the next two lanes are PCR samples from the parental strains B (higher band at 198 bp) and D (lowest band at 176 bp). Lanes 4 to 25 are the F2 samples. The bottom band—or bands, in the case of heterozygotes—define the genotype of each animal. The bands at the top of the figure are caused by DNA retained in the pipetting wells. Figure from work by G. Zhou (Zhou & Williams, 1997).

Questions and Answers

1. Is there any particular reason to use Ficoll rather than sucrose in the PCR? ANSWER: No. We initially used sucrose, but Ficoll works slightly better. (I think that it may be a bit heavier than 60% sucrose, but would need to verify this.)

2. Does cresol red run with the DNA of a particular size in a 3% metaphor gel? (or maybe it runs the wrong way). ANSWER: The cresol red will run close to the primer dimer (less than 75 bp).

3. What Tq do you use? ANSWER: We use Promega Taq. We use the cheaper grade.

4. Metaphor agarose is very expensive. Do you reuse it? And if not, why not? ANSWER: Yes, we reuse the MetaPhor 3 to 7 times (see notes above on "Economizing").

5. Do you have any sense of how much resolution is lost if the gel is run at room temp at half the speed you use? ANSWER: When we run the gels cold and at high voltage as described above, we can consistently resolve 6 bp, sometimes 4 bp, and rarely 2 bp. If the gel runs hot, then bands will often be bowed and distorted and have a lower intensity. The quality of bands depends to a great extent on the particular primer pair. With a good primer pair you certainly could run at room temperature or higher at a lower voltage.

6. Do you know of any reasons why the method would not be useful for multiplex PCR? ANSWER: We have multiplexed using the same protocol. You will need to test and optimize primer sets and you will probably need a base-pair gap of 10 bp. This usually involves changeing relative primer concentrations, with higher concentrations used for those primers that amplify the larger product.

7. Can I cast the Metaphor slab and leave it out overnight? Yes, but refrigerate the slab overnight at 4 degrees.

8. What is the cost per genotype as described in this protocol?

  • DNA extraction is a fixed one-time cost. To extract enough DNA for 1000 reactions or more using the protocol listed above is about $0.40/case (DNA from spleen).

    Our costs are as follows per single reaction of 10 &#micro;l:

    1. Cost for Taq: $0.06
    2. Cost of PCR primers from Res Gen: $0.02
    3. Cost of dNTP: $0.02
    4. Cost of gel (includes ecomony achieved by recycling): $0.05
    5. Cost of Polaroid photo (film cost divided by 96 lanes): $0.01
    6. Cost of plastic ware: $0.07

    Total genotype cost is therefore $0.23 + DNA preparation/number of genotypes per case.

    Labor expense: We assume that one full time technician can carry out approximately 6 x 96 well plates per day. This includes all data entry. To achieve this thoughput does require a 96-channel Robinson pipette station. Therefore to compute total expense you will need to divide the technician salary by 576. Assume total technical cost of 33K/year (including fringe etc, assume 48 weeks per year and 5/days per week) = $140/day. This equals about $0.25 per genotype per day.

    The cost of personnel and supplies is therefore approximately matched using our methods.

    Ways to economize further. 1. Make Taq; 2. Use even cheaper DNA extraction method.

    For a current evaluation of genotyping methods see: Weber JL, Broman KW (2000) Genotyping for human whole-genome scans: past, present, and future. Adv in Genet 42:77–96.


    Since 15 Oct 1998
    Revised 21 Jan 2001



    Neurogenetics at University of Tennessee Health Science Center

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