Gene Synthsis Error Removal

Cel-1 Protocol

Cel-1: Can be purchased from Transgenomic, Inc.

1. Run PCR gene synthesis reaction on an agarose gel, purify desired band using Qiagen gel extraction kit. Elute DNA with 50 ul TE.

2. Denature purified DNA at 95 oC for 10 min, anneal at 0.1 oC per second for 10 min in a buffer containing 100 mM Tris-HCl, pH 8.0, 1 mM EDTA and 1 M NaCl.

3. Purify DNA using Qiagen PCR purification kit.

4. Add buffer (10 mM Tris-HCl, pH 8.8, 75 mM KCl, 1.5 mM MgSO4) to DNA in 45 ul, add 5 ul of mis-match specific endonuclease CEL1 nuclease from Transgenomic, Inc. Incubate at 40 oC for 60 min.

5. Run digested DNA on agarose gel, cut out "error-free" CEL1 resistant band and purify DNA using Qiagen gel extraction kit

SURVEYOR Mutation Detection Kit For Standard Gel Electrophoresis
Catalog No. Quantity

706025 25 Reactions
706020 100 Reactions
706021 1,000 Reactions

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12325 Emmet Street
Omaha, NE 68164
Phone: (888) 233-WAVE / (888) 233-9283
Fax: (402) 452-5453
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TaqMuts Protocol

1. After PCR, heat PCR product at 95°C for 2 minutes and then gradually ramping down the temperature to room temperature over the course of 20-30 minute.

2. Purify PCR product using gel extraction kit or PCR purification kit.

3. Add Recombinant MBP-TaqMuts (from Excellgen), add binding buffer (50 mM NaCl, 10 mM Tris pH 8, and 8 mM MgCl2).

4. Incubate at 60 oC for 10 min.

5. Run on agarose gel using GRGreen DNA dye. The error-containing MutS-bound DNA will migrate slower than "MutS-filtered" DNA fragment. This is so-called gel-mobility shift.

6. Cut out disired band and purify DNA using Qiagen gel purification kit.