http://www.mountsinai.org/Education/School%20of%20Medicine
home.html
sorianosoriano.htmlshapeimage_3_link_0
protocolssorianopro.htmlshapeimage_4_link_0
ES cell culturesorianoescc.htmlshapeimage_5_link_0
ES screening by PCRsorianoespcr.htmlshapeimage_7_link_0
southern blotsorianoblot.htmlshapeimage_8_link_0
ES screening by southernssorianoess.htmlshapeimage_9_link_0
x-gal stainingsorianoxgs.htmlshapeimage_10_link_0
http://gallery.me.com/drbsinai#100039&bgcolor=black&view=mosaic&sel=0
 

Soriano Lab - Protocols

3sorianopro.html

PCR Protocol

    We use a different PCR buffer system that requires no magnesium optimization. In our hands this is much more sensitive than the standard PCR buffer. The method we use was first described by Jane Gitschier's group (NEW ENGLAND J. MED. 1987. 317, 985-990)

    Although most PCRs do not need a hot start, this may improve specificity and yield. After the first denaturation step (93°C for 2 min), add 5 μl of Taq/dNTP mix to each reaction while holding them at 82°C. Proceed to thermal cycling. Annealing temperatures are typically 55°C ±5°C (needs to be determined), extension temperature is 65°C.

Sample prep

    DNA samples can be made as follows: Take up cells (e.g.. 1/5 of a ES colony, or a ~l mm tail or toe clip) in a 0.65 ml tube. Add 10 μl (for cells) or 40 μl (for a tail piece) of proteinase K buffer (l x MGB without gelatin, with 1% 2-ME and 0.5% Triton X-100; proteinase K at a final concentration of 400 μg/ml). Incubate at 55°C for 60 min., heat-denature at 95°C for 5 min. (optional for hot start). Use 1-5μl per PCR reaction.

Reagents:              PCR Lysis Buffer

                            10.08 ml of MQ water

                            01.20 ml of 10X GB

                            00.60 ml of 10% Tx-100 (Triton X-100)

                            00.12 ml of 2-ME (optional)

                            12.00 ml final volume (store in frig.)

                            Add proteinase K just prior to use (40μg/ml)


Gitschier's Buffer (GB) 10X recipe given:                                                 (final concentration)

                                                                                 

                            3.35 mL of 2 M Tris pH=8.8                                       670 mM Tris pH=8.8

                            1.66 mL of 1 M (NH4)2SO4                                        166 mM Ammonium Sulfate

                            1.34 mL of 0.5 M MgCl2                                             67 mM MgCl2

                            3.65 mL MQ H2O

                            10.0 mL final volume (store in freezer)