Editing Gene Synthesis by PCR (section)

== Protocol 2 ==

1. Design sequence using Invitrogen's gene synthsis (GeneArt) web software<br/>Sign up an account, and do codon optimization for free

Alternatively, design gene sequence using IDT's web site, [https://www.idtdna.com/pages/products/genes/custom-gene-synthesis https://www.idtdna.com/pages/products/genes/custom-gene-synthesis], it is also free

Design 500 to 800 bp fragments for oligo synthsis, for example Taq Ploymerase can be made by gene synthesis of 4 PCR fragments

[http://www.ncbi.nlm.nih.gov/nuccore/155128 http://www.ncbi.nlm.nih.gov/nuccore/155128]

atgaggggga tgctgcccct ctttgagccc aagggccggg tcctcctggt ggacggccac<br/>181 cacctggcct accgcacctt ccacgccctg aagggcctca ccaccagccg gggggagccg<br/>241 gtgcaggcgg tctacggctt cgccaagagc ctcctcaagg ccctcaagga ggacggggac<br/>301 gcggtgatcg tggtctttga cgccaaggcc ccctccttcc gccacgaggc ctacgggggg<br/>361 tacaaggcgg gccgggcccc cacgccggag gactttcccc ggcaactcgc cctcatcaag<br/>421 gagctggtgg acctcctggg gctggcgcgc ctcgaggtcc cgggctacga ggcggacgac<br/>481 gtcctggcca gcctggccaa gaaggcggaa aaggagggct acgaggtccg catcctcacc<br/>541 gccgacaaag acctttacca gctcctttcc gaccgcatcc acgtcctcca ccccgagggg<br/>601 tacctcatca ccccggcctg gctttgggaa aagtacggcc tgaggcccga ccagtgggcc<br/>661 gactaccggg ccctgaccgg ggacgagtcc gacaaccttc ccggggtcaa gggcatcggg<br/>721 gagaagacgg cgaggaagct tctggaggag tgggggagcc tggaagccct cctcaagaac<br/>781 ctggaccggc tgaagcccgc catccgggag aagatcctgg cccacatgga cgatctgaag<br/>841 ctctcctggg acctggccaa ggtgcgcacc gacctgcccc tggaggtgga cttcgccaaa<br/>901 aggcgggagc ccgaccggga gaggcttagg gcctttctgg agaggcttga gtttggcagc<br/>961 ctcctccacg agttcggcct tctggaaagc cccaaggccc tggaggaggc cccctggccc<br/>1021 ccgccggaag gggccttcgt gggctttgtg ctttcccgca aggagcccat gtgggccgat<br/>1081 cttctggccc tggccgccgc cagggggggc cgggtccacc gggcccccga gccttataaa<br/>1141 gccctcaggg acctgaagga ggcgcggggg cttctcgcca aagacctgag cgttctggcc<br/>1201 ctgagggaag gccttggcct cccgcccggc gacgacccca tgctcctcgc ctacctcctg<br/>1261 gacccttcca acaccacccc cgagggggtg gcccggcgct acggcgggga gtggacggag<br/>1321 gaggcggggg agcgggccgc cctttccgag aggctcttcg ccaacctgtg ggggaggctt<br/>1381 gagggggagg agaggctcct ttggctttac cgggaggtgg agaggcccct ttccgctgtc<br/>1441 ctggcccaca tggaggccac gggggtgcgc ctggacgtgg cctatctcag ggccttgtcc<br/>1501 ctggaggtgg ccgaggagat cgcccgcctc gaggccgagg tcttccgcct ggccggccac<br/>1561 cccttcaacc tcaactcccg ggaccagctg gaaagggtcc tctttgacga gctagggctt<br/>1621 cccgccatcg gcaagacgga gaagaccggc aagcgctcca ccagcgccgc cgtcctggag<br/>1681 gccctccgcg aggcccaccc catcgtggag aagatcctgc agtaccggga gctcaccaag<br/>1741 ctgaagagca cctacattga ccccttgccg gacctcatcc accccaggac gggccgcctc<br/>1801 cacacccgct tcaaccagac ggccacggcc acgggcaggc taagtagctc cgatcccaac<br/>1861 ctccagaaca tccccgtccg caccccgctt gggcagagga tccgccgggc cttcatcgcc<br/>1921 gaggaggggt ggctattggt ggccctggac tatagccaga tagagctcag ggtgctggcc<br/>1981 cacctctccg gcgacgagaa cctgatccgg gtcttccagg aggggcggga catccacacg<br/>2041 gagaccgcca gctggatgtt cggcgtcccc cgggaggccg tggaccccct gatgcgccgg<br/>2101 gcggccaaga ccatcaactt cggggtcctc tacggcatgt cggcccaccg cctctcccag<br/>2161 gagctagcca tcccttacga ggaggcccag gccttcattg agcgctactt tcagagcttc<br/>2221 cccaaggtgc gggcctggat tgagaagacc ctggaggagg gcaggaggcg ggggtacgtg<br/>2281 gagaccctct tcggccgccg ccgctacgtg ccagacctag aggcccgggt gaagagcgtg<br/>2341 cgggaggcgg ccgagcgcat ggccttcaac atgcccgtcc agggcaccgc cgccgacctc<br/>2401 atgaagctgg ctatggtgaa gctcttcccc aggctggagg aaatgggggc caggatgctc<br/>2461 cttcaggtcc acgacgagct ggtcctcgag gccccaaaag agagggcgga ggccgtggcc<br/>2521 cggctggcca aggaggtcat ggagggggtg tatcccctgg ccgtgcccct ggaggtggag<br/>2581 gtggggatag gggaggactg gctctccgcc aaggagtga

Nde1<br/>CATATG

ATGAGGGGGATGCTGCCCCTCTTTGAGCCCAAGGGCCGGGTCCTCCTGGTGGACGGCCACCACCTGGCCTACCGCACCTTCCACGCCCTGAAGGGCCTCACCACCAGCCGGGGGGAGCCGGTGCAGGCGGTCTACGGCTTCGCCAAGAGCCTCCTCAAGGCCCTCAAGGAGGACGGGGACGCGGTGATCGTGGTCTTTGACGCCAAGGCCCCCTCCTTCCGCCACGAGGCCTACGGGGGGTACAAGGCGGGCCGGGCCCCCACGCCGGAGGACTTTCCCCGGCAACTCGCCCTCATCAAGGAGCTGGTGGACCTCCTGGGGCTGGCGCGCCTCGAGGTCCCGGGCTACGAGGCGGACGACGTCCTGGCCAGCCTGGCCAAGAAGGCGGAAAAGGAGGGCTACGAGGTCCGCATCCTCACCGCCGACAAAGACCTTTACCAGCTCCTTTCCGACCGCATCCACGTCCTCCACCCCGAGGGGTACCTCATCACCCCGGCCTGGCTTTGGGAAAAGTACGGCCTGAGGCCCGACCAGTGGGCCGACTACCGGGCCCTGACCGGGGACGAGTCCGACAACCTTCCCGGGGTCAAGGGCATCGGGGAGAAGACGGCGAGGAAGCTTCTGGAGGAG<br/>(GGAGAAGACGGCGAGGAAGCTTCTGGAGGAG)TGGGGGAGCCTGGAAGCCCTCCTCAAGAACCTGGACCGGCTGAAGCCCGCCATCCGGGAGAAGATCCTGGCCCACATGGACGATCTGAAGCTCTCCTGGGACCTGGCCAAGGTGCGCACCGACCTGCCCCTGGAGGTGGACTTCGCCAAAAGGCGGGAGCCCGACCGGGAGAGGCTTAGGGCCTTTCTGGAGAGGCTTGAGTTTGGCAGCCTCCTCCACGAGTTCGGCCTTCTGGAAAGCCCCAAGGCCCTGGAGGAGGCCCCCTGGCCCCCGCCGGAAGGGGCCTTCGTGGGCTTTGTGCTTTCCCGCAAGGAGCCCATGTGGGCCGATCTTCTGGCCCTGGCCGCCGCCAGGGGGGGCCGGGTCCACCGGGCCCCCGAGCCTTATAAAGCCCTCAGGGACCTGAAGGAGGCGCGGGGGCTTCTCGCCAAAGACCTGAGCGTTCTGGCCCTGAGGGAAGGCCTTGGCCTCCCGCCCGGCGACGACCCCATGCTCCTCGCCTACCTCCTGGACCCTTCCAACACCACCCCCGAGGGGGTGGCCCGGCGCTACGGCGGGGAGTGGACGGAGGAGGCGGGGGAGCGGGCCGCCCTTTCCGAGAGGCTCTTCGCCAACCTGTGGGGGAGGCTTG<br/>(GAGAGGCTCTTCGCCAACCTGTGGGGGAGGCTTG)AGGGGGAGGAGAGGCTCCTTTGGCTTTACCGGGAGGTGGAGAGGCCCCTTTCCGCTGTCCTGGCCCACATGGAGGCCACGGGGGTGCGCCTGGACGTGGCCTATCTCAGGGCCTTGTCCCTGGAGGTGGCCGAGGAGATCGCCCGCCTCGAGGCCGAGGTCTTCCGCCTGGCCGGCCACCCCTTCAACCTCAACTCCCGGGACCAGCTGGAAAGGGTCCTCTTTGACGAGCTAGGGCTTCCCGCCATCGGCAAGACGGAGAAGACCGGCAAGCGCTCCACCAGCGCCGCCGTCCTGGAGGCCCTCCGCGAGGCCCACCCCATCGTGGAGAAGATCCTGCAGTACCGGGAGCTCACCAAGCTGAAGAGCACCTACATTGACCCCTTGCCGGACCTCATCCACCCCAGGACGGGCCGCCTCCACACCCGCTTCAACCAGACGGCCACGGCCACGGGCAGGCTAAGTAGCTCCGATCCCAACCTCCAGAACATCCCCGTCCGCACCCCGCTTGGGCAGAGGATCCGCCGGGCCTTCATCGCCGAGGAGGGGTGGCTATTGGTGGCCCTGGACTATAGCCAGATAGAGCTCAGGGTGCTGGCCCACCTCTCCGGCGACGAGAACCTGATCCGGGTC<br/>(CCTCTCCGGCGACGAGAACCTGATCCGGGTC)TTCCAGGAGGGGCGGGACATCCACACGGAGACCGCCAGCTGGATGTTCGGCGTCCCCCGGGAGGCCGTGGACCCCCTGATGCGCCGGGCGGCCAAGACCATCAACTTCGGGGTCCTCTACGGCATGTCGGCCCACCGCCTCTCCCAGGAGCTAGCCATCCCTTACGAGGAGGCCCAGGCCTTCATTGAGCGCTACTTTCAGAGCTTCCCCAAGGTGCGGGCCTGGATTGAGAAGACCCTGGAGGAGGGCAGGAGGCGGGGGTACGTGGAGACCCTCTTCGGCCGCCGCCGCTACGTGCCAGACCTAGAGGCCCGGGTGAAGAGCGTGCGGGAGGCGGCCGAGCGCATGGCCTTCAACATGCCCGTCCAGGGCACCGCCGCCGACCTCATGAAGCTGGCTATGGTGAAGCTCTTCCCCAGGCTGGAGGAAATGGGGGCCAGGATGCTCCTTCAGGTCCACGACGAGCTGGTCCTCGAGGCCCCAAAAGAGAGGGCGGAGGCCGTGGCCCGGCTGGCCAAGGAGGTCATGGAGGGGGTGTATCCCCTGGCCGTGCCCCTGGAGGTGGAGGTGGGGATAGGGGAGGACTGGCTCTCCGCCAAGGAGTGA

Sal1<br/>GTCGAC

2. Design Oligos for each fragment using NIH DNAWorks software [http://helixweb.nih.gov/dnaworks/ http://helixweb.nih.gov/dnaworks/]

Copy and paste DNA sequence to the website, use 50 to 60 nt, do not need to change any other parameters<br/>Select the oligo design (TRIAL) with the lowest Score (with as few # of misprime as possible).<br/>Make sure there are 30 nt overlaps between each PCR fragment.

3. Order oligos from IDT (on plate, should be 8 cents per base).

4. Make oligo stock solution and mixture<br/>Mix: take 1 ul from each 10 uM oligo stock in water.

5. 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 10X buffer<br/>1 ul of 10 mM dNTP<br/>Water to 25 ul

6. PCR

mix two PCR reactions, add two outer oligo and continue PCR 25 cycles.

7. Run PCR product on gel, purify using Qiagen gel extraction kit

8. Join small fragments with Gibson mix.

9. Purify assembled product on gel.

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

11. Verify clone by sequencing (Macrogen, $3.5 per reaction).<br/>
[[Category:Cloning]] [[Category:Protocols]]