Reaction mix and thermocycling conditions:

Unless otherwise noted, all primers for PCR used in the Roopenian lab and listed on our web site have been designed to work at an annealing temperature of 60oC.  SSLP markers typically require 40 cycles. Most of our other primers give more robust results and only require 35 cycles.

Tom’s Master Mix (without primers or DNA)

 
   1x                2400x

10.18 µl          24.5 ml               18 MOhm H2O
  1.5   µl            3.6 ml               30% Sucrose/Cresol Red1
  1.5   µl            3.6 ml               Eppendorf 10X buffer w/ 25mM MgCl2
  0.12 µl           288 µl                25 mM dNTPs
  0.1   µl           200 µl (1 vial)    Eppendorf Hot Master taq polymerase

Totals:

13.4   µl        32.16 ml              Total Tom’s Master Mix

Master Mix with Primer
  

     1x                10x (as an example)

13.4 µl             134 ml             Tom’s Master Mix
  0.6 µl                 6 ml             10 uM primer (either mixed primers or separate primers)

Load 14 ul Master Mix with Primer per well.  Add 1 ul DNA.

Thermocycling:

  1. 95oC for 1 minute
  2. 95oC for 15 seconds
  3. 60oC for 20 seconds
  4. 70oC for 30 seconds
    Repeat steps 2 to 4 for 40 cycles
  5. 4oC/End

 

Our protocols to isolate DNA in 96-well plates from:

            Peripheral Blood
            Tail snips

Note that making a large master mix stock in advance allows me to use only .42 Units of polymerase per reaction.  I have stored portions of 2400X Master Mix for extended periods (>4 weeks) at 4oC without noticeable deterioration of PCR quality.

Note also that since the reaction mix already includes sucrose and Cresol Red loading dye, reactions are ready to load on a gel as soon as the thermocycling is completed, without the need for a separate dye loading step.  I have tried reaction mixes with other dyes, such as Bromphenyl Blue, but Cresol Red is the only one I have found which does not impede the PCR.  If you do not wish to include the Sucrose/Cresol Red in you master mix, simply replace with H2O.

For particular genotyping assays, I combine appropriate amounts of  Tom’s Master Mix and primer mix (I tend to store all of my working stocks of primers as 10 µM forward and reverse mixed rather then separated) such that I can then distribute 14 µl of mix including primers (13.4 µl Master Mix with 0.6 µl of 10 uM primer mix) to the reaction wells and then add 1 µl of DNA per well.  Note that this reaction mix is fairly forgiving.  If you store F and R primers separately, you may add 0.6 ul of each to the master mix + primer combination without the need to reduce the volume of Master Mix.

 

Gels:

SSLP genotyping is done using a 0.7% Agarose2, 1.5% Synergel3 gel. For an example 100 ml gel, first combine 0.7 grams Agarose and 1.5 grams Synergel in a dry flask.  Add a little 70% ethanol and mix the powders into a slurry.  For a running buffer, we use TAE.  Per liter of working stock TAE required, combine 20 ml 50x TAE and 15 µl of 10 mg/ml Ethiduium Bromide in distilled H2O.  For the gel, add 100 ml of 1x TAE with Ethidium Bromide to the agarose slurry. Heat to a boil.

Genotyping for transgenics and knockouts can be done on the same gel as is used for SSLP, but can also be done on a gel using 1.2& Agarose and no Synergel.

 

130% Sucrose/Cresol Red:

     30 grams Sucrose
     ~1 gram Cresol Red
   100 ml  18 MOhm H2O (start volume)
   Sterile filter.  Store at 4oC.

2Agarose we use is GenePure LE Quci Dissolve Agarose from ISC BioExpress, 500 grams, Cat. No. E-3119-500.

3Synergel Agarose Clarifying Additive is from Diversified Biotech, 100 grams, Cat. No. SYN-100.

If any questions, contact Tom Sproule at tom.sproule@jax.org.