Gene Synthesis by PCR

Gene Synthesis by PCR using oligo as template

Oligo design

analyse DNA sequence using http://tools.neb.com/NEBcutter2. Select a pair of common restriction enzymes, such as BamH1, EcoR1, Xho1. Add enzyme sequences to both ends. Add 6 nt before 5' and after 3' end.

Use a cloning vector such pET28, pET30, pCDNA3.

Design 55mer to 60mer oligos with 15~20 nt sequence overlap, Tm: ~55 oC.

Software: http://helixweb.nih.gov/dnaworks/

select the least mismatch, lowest score, and the length of the first primer and the last primer is not too much different.

copy oligos to text editor, remove numbers, copy oligos to oligo order spreadsheet.

Preparation of Oligo Solution

for 96 well plates:

1. add TE to each well to make 100 uM, for example, add 132 ul TE to the well with 13.2 nmole oligo. This is 100 uM stock

2. let the plate sit on bench for 15 min.

3, add 90 ul of ddH2O to another plate, transfer 10 ul from from 100 uM stock, mix. This is 10 uM stock. Take out 1 ul from each well for gene synthsis mix. For PCR primer take out 15~20 ul from the well. 

4. Label the plate with freeze-resistant lable. stock inside -20 oC freezer.

5. Store the 100 uM stock plate inside -80 oC freezer

if you need to use more oligo solutions, take the 10 uM plate out and thaw the solution at room templature or inside a 37 oC incubater oven.

1st round PCR

~ 12 oligos for each ~500 bp block

mix 10 to 24 of oligos: take 1 ul of 10 uM oligo from each tube, put them in one tube.

if the number of oligos is <10, add water to 10 ul.

Take 0.5 or 1 ul of oligo mixture for 25 ul PCR.

PCR
1 ul of 10 uM outer oligos for 25 ul PCR, 1 ul 10 uM dNTP
1 Units of  Phusion.

2 min at 95 oC

10 cycles:
30 s at 95 oC
30 s at 55 oC
1 min at 72 oC

1 Step Gene synthesis for < 800 bp: continue another 15 cycles.

2nd round PCR (for >800bp)

1~2 ul of  1st round PCR
1 ul of 10 uM outer oligos for 25 ul PCR.

94 oC for 2 min, then add 1 U of Phusion.

then 25 cycles:
94 oC for 30 s
55 oC for 30 s
72 oC for  <4 kb gene, 1kb/min

or 68 oC  for >4 kb gene, 1 kb/ 2 min.

final: 72 oc for 5 min

Run agarose gel, cut out band and purify using spin column, digest with restriction enzymes, ligate to a cloning vector, transform Top10

Verify gene synthesis product by restriction enzyme digestion and sequencing.

Protocol 2

1. Design sequence using Invitrogen's gene synthsis (GeneArt) web software
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, 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

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

Nde1
CATATG

ATGAGGGGGATGCTGCCCCTCTTTGAGCCCAAGGGCCGGGTCCTCCTGGTGGACGGCCACCACCTGGCCTACCGCACCTTCCACGCCCTGAAGGGCCTCACCACCAGCCGGGGGGAGCCGGTGCAGGCGGTCTACGGCTTCGCCAAGAGCCTCCTCAAGGCCCTCAAGGAGGACGGGGACGCGGTGATCGTGGTCTTTGACGCCAAGGCCCCCTCCTTCCGCCACGAGGCCTACGGGGGGTACAAGGCGGGCCGGGCCCCCACGCCGGAGGACTTTCCCCGGCAACTCGCCCTCATCAAGGAGCTGGTGGACCTCCTGGGGCTGGCGCGCCTCGAGGTCCCGGGCTACGAGGCGGACGACGTCCTGGCCAGCCTGGCCAAGAAGGCGGAAAAGGAGGGCTACGAGGTCCGCATCCTCACCGCCGACAAAGACCTTTACCAGCTCCTTTCCGACCGCATCCACGTCCTCCACCCCGAGGGGTACCTCATCACCCCGGCCTGGCTTTGGGAAAAGTACGGCCTGAGGCCCGACCAGTGGGCCGACTACCGGGCCCTGACCGGGGACGAGTCCGACAACCTTCCCGGGGTCAAGGGCATCGGGGAGAAGACGGCGAGGAAGCTTCTGGAGGAG
(GGAGAAGACGGCGAGGAAGCTTCTGGAGGAG)TGGGGGAGCCTGGAAGCCCTCCTCAAGAACCTGGACCGGCTGAAGCCCGCCATCCGGGAGAAGATCCTGGCCCACATGGACGATCTGAAGCTCTCCTGGGACCTGGCCAAGGTGCGCACCGACCTGCCCCTGGAGGTGGACTTCGCCAAAAGGCGGGAGCCCGACCGGGAGAGGCTTAGGGCCTTTCTGGAGAGGCTTGAGTTTGGCAGCCTCCTCCACGAGTTCGGCCTTCTGGAAAGCCCCAAGGCCCTGGAGGAGGCCCCCTGGCCCCCGCCGGAAGGGGCCTTCGTGGGCTTTGTGCTTTCCCGCAAGGAGCCCATGTGGGCCGATCTTCTGGCCCTGGCCGCCGCCAGGGGGGGCCGGGTCCACCGGGCCCCCGAGCCTTATAAAGCCCTCAGGGACCTGAAGGAGGCGCGGGGGCTTCTCGCCAAAGACCTGAGCGTTCTGGCCCTGAGGGAAGGCCTTGGCCTCCCGCCCGGCGACGACCCCATGCTCCTCGCCTACCTCCTGGACCCTTCCAACACCACCCCCGAGGGGGTGGCCCGGCGCTACGGCGGGGAGTGGACGGAGGAGGCGGGGGAGCGGGCCGCCCTTTCCGAGAGGCTCTTCGCCAACCTGTGGGGGAGGCTTG
(GAGAGGCTCTTCGCCAACCTGTGGGGGAGGCTTG)AGGGGGAGGAGAGGCTCCTTTGGCTTTACCGGGAGGTGGAGAGGCCCCTTTCCGCTGTCCTGGCCCACATGGAGGCCACGGGGGTGCGCCTGGACGTGGCCTATCTCAGGGCCTTGTCCCTGGAGGTGGCCGAGGAGATCGCCCGCCTCGAGGCCGAGGTCTTCCGCCTGGCCGGCCACCCCTTCAACCTCAACTCCCGGGACCAGCTGGAAAGGGTCCTCTTTGACGAGCTAGGGCTTCCCGCCATCGGCAAGACGGAGAAGACCGGCAAGCGCTCCACCAGCGCCGCCGTCCTGGAGGCCCTCCGCGAGGCCCACCCCATCGTGGAGAAGATCCTGCAGTACCGGGAGCTCACCAAGCTGAAGAGCACCTACATTGACCCCTTGCCGGACCTCATCCACCCCAGGACGGGCCGCCTCCACACCCGCTTCAACCAGACGGCCACGGCCACGGGCAGGCTAAGTAGCTCCGATCCCAACCTCCAGAACATCCCCGTCCGCACCCCGCTTGGGCAGAGGATCCGCCGGGCCTTCATCGCCGAGGAGGGGTGGCTATTGGTGGCCCTGGACTATAGCCAGATAGAGCTCAGGGTGCTGGCCCACCTCTCCGGCGACGAGAACCTGATCCGGGTC
(CCTCTCCGGCGACGAGAACCTGATCCGGGTC)TTCCAGGAGGGGCGGGACATCCACACGGAGACCGCCAGCTGGATGTTCGGCGTCCCCCGGGAGGCCGTGGACCCCCTGATGCGCCGGGCGGCCAAGACCATCAACTTCGGGGTCCTCTACGGCATGTCGGCCCACCGCCTCTCCCAGGAGCTAGCCATCCCTTACGAGGAGGCCCAGGCCTTCATTGAGCGCTACTTTCAGAGCTTCCCCAAGGTGCGGGCCTGGATTGAGAAGACCCTGGAGGAGGGCAGGAGGCGGGGGTACGTGGAGACCCTCTTCGGCCGCCGCCGCTACGTGCCAGACCTAGAGGCCCGGGTGAAGAGCGTGCGGGAGGCGGCCGAGCGCATGGCCTTCAACATGCCCGTCCAGGGCACCGCCGCCGACCTCATGAAGCTGGCTATGGTGAAGCTCTTCCCCAGGCTGGAGGAAATGGGGGCCAGGATGCTCCTTCAGGTCCACGACGAGCTGGTCCTCGAGGCCCCAAAAGAGAGGGCGGAGGCCGTGGCCCGGCTGGCCAAGGAGGTCATGGAGGGGGTGTATCCCCTGGCCGTGCCCCTGGAGGTGGAGGTGGGGATAGGGGAGGACTGGCTCTCCGCCAAGGAGTGA

Sal1
GTCGAC

2. Design Oligos for each fragment using NIH DNAWorks software 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
Select the oligo design (TRIAL) with the lowest Score (with as few # of misprime as possible).
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
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
0.5 ul oligo mix
0.5 ul polymerase enzyme
5 ul of 10X buffer
1 ul of 10 mM dNTP
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).