Editing Gene-Synthesis-Error Correction

1. Make oligo stock solution and mixture.

oligo mix: take 1 ul from each 10 uM oligo stock in water.

2. Template generation PCR: 5 cycles using Phusion hot start DNA polymerase or Kapa HiFi<br/>0.5 ul oligo mix<br/>0.5 ul polymerase enzyme<br/>5 ul of&nbsp; 5X buffer<br/>1 ul of 10 mM dNTP<br/>Water to 25 ul

3. After template generation PCR, heat the reaction at 95 oC for 10 min, cool to 85 oC at 2 oC/s and hold for1 min. then cool down to 25 oC at a rate of 0.3 oC, holding for 1 min at every 10 oC interval.

<br/>4. Add 0.5 ul of Surveyor nuclease and 0.5 ul enhancer (Ligase, T4 DNA ligase?), incubated at 42 oC for 20 min

this patent using&nbsp;Surveyor nuclease plus&nbsp; Exonuclease III: [https://www.google.com/patents/WO2013116771A1 https://www.google.com/patents/WO2013116771A1]

or&nbsp;&nbsp;T4 endonuclease , or&nbsp;Endonuclease V

or&nbsp;T4 endonuclease + Exonuclease III, or&nbsp;Endonuclease V + Exonuclease III

or T7 endonuclease 1

but not as effective as SURVEYOR nuclease alone

<br/>5. Add 2 outer primers to amplify gene of interest. Run PCR for 25 cycles.

6. Separate PCR product on agarose gel. Purify desired band using Qiagen gel extraction kit. Elute DNA with 20 ul water.

7. Digest with restriction enzyme and clone into a vector.

== CorrectASE™ Error Correction Protocol (Thermofisher) ==

<br/>CorrectASE:&nbsp; Surveyor nuclease + enhancer (Ligase, maybe T4 DNA ligase)

$294/50 reaction&nbsp;<br/>https://www.thermofisher.com/order/catalog/product/A14972

1. Measure oligo DNA concentration&nbsp;

2. In a PCR tube, dilute the DNA to 20–25 ng/µL in 1X CorrectASE™ Reaction Buffer&nbsp;<br/>Final volume is 50 µL&nbsp;

3. Denature and re-anneal the diluted DNA to create mismatches using the following conditions:

98°C for 2 minutes<br/>4°C for 5 minutes<br/>37°C for 5 minutes<br/>4°C hold

4. Place the re-annealed DNA dilution on ice and transfer 10 µL to a separate PCR tube.

5. To 10 µL of the re-annealed DNA dilution, add 1 µL of CorrectASE™ enzyme and incubate at 25°C for 1 hour.

6. After the incubation, immediately place the reaction mix on ice

7. Proceed immediately to the final PCR amplification step (Do not stop).


[[Category:Protocols]]