Keating:Gene Synthesis

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Gene Synthesis Protocol

I.) Choose protein sequence to synthesize, flanking tags, restriction sites and or clamping regions A.) For Gene Synthesis into pTWIN1 include a 5` BamHI site and a 3` NotI site II.) Use DNAworks ( http://molbio.info.nih.gov/dnaworks/ ) to develop oligos to use for the synthesis A.) Refer to (Hoover and Lubkowski, Nucleic Acids Research, 2002, Vol. 30, No. 10) and DNAWorks Guide at (http://molbio.info.nih.gov/dnaworks/dnaworks_help.html ) for any questions B.) Be sure to screen the gene synthesis design to exclude BamHI and NotI from the gene and in proper frame C.) Change the Mg Concentration to 1.5mM and change the oligo concentration to 2.5E-6M (2500 nM) D.) One can also play with other parameters to optimize the oligo length (<40), avoid direct repeats and lower the overall score E.) Keep a printout of the DNAWorks output email for reference III.) After running the synthesis by Amy order oligos (25mM for <42 and 100mM for >42) IV.) Spin oligos down and re-suspend in 100μl filtered water, then make dilutions (1:200 for 25mM and 1:800 for 100mM) to test the purity and concentrations of the oligos V.) Take OD spectra from 280 to 260. The ratio of OD260:OD280 = purity, ideally (pure) the ratio is 1.8. To calculate the concentration into pMol/μl = OD260(35μg/ml)(MW in μmol/μg)(dilution factor)(1000). Note 1000 comes from (1000nmol/μmol)(1ml/1000μl)(1000pmol/nmol) VI.) Make a cocktail of the oligos at 2.5μM= 2.5pmol/μl each, a cocktail of dNTP’s at 10mM each, and a cocktail of the first and last oligos (oligos used at the 5` end of each strand in gene synthesis) at 50μM = 50pmol/μl each VII.) Set up 2-3 PCR reactions #1/gene on ice; add sequentially 42.25μl filtered water, .5μl pooled oligos, 1.25μl dNTP mix, 5μl 10X turbo polymerase buffer, and 1μl Pfu turbo Hotstart polymerase, for a 50 μl reaction, lastly add 50μl mineral oil very slowly down the side of the PCR tube


VIII.) PCR reaction #1 program default is 95° C for 2 min., 30 cycles of; [95° C denaturation for 30 sec., ° C annealing 30 sec., (Whatever DNAWorks calls for 62° C by default), 72° C extension1 min.], and a final 72° C extension for 2 min., 4°C unlimited time IX.) Run PCR product on a high percent gel and look for a band or a smear around the expected product size, take a picture and cut out the band or smear X.) Gel purify the band(s) and use the DNA as the template in PCR reaction #2. Do 2-3 PCR reactions/gene, on ice add 41.2μl filtered water, 1.25μl dNTP mix (10mM each), .6μl first and last oligo cocktail, 5 μl 10X turbo polymerase buffer, 1μl turbo polymerase, and 1μl PCR reaction #1 product sequentially for a single 50 μl reaction, Slowly add 50μl mineral oil XI.) PCR reaction #2 program; 95°C hot start 2 min., 30 cycles of; [95°C denaturation 30 sec., °C 30 sec. annealing (temp. varies depending on primers=first and last oligos cocktail, but 55°C can be used a s default), 72°C extension 1 min.], then a 72°C incubation for 2 min., and 4°C final step for unlimited time XII.) Run PCR product on a gel and look for a band corresponding to the size of the expected product, excise the band and gel purify it. XIII.) Cut the synthesized and purified gene and the cloning vector with the appropriate restriction enzymes depending on the vector. For pTWIN1 use BamHI and Not1, NEB suggests a sequential digestion for these enzymes XIV.) Ligate the gene into the vector using the rapid ligation kit and the lab protocol, then transform into XL1-Blue cells following the general lab protocol. PTWIN is Amp resistant XV.) Pick 3 clones from each plate and grow overnights, mini-prep, send for sequencing XVI.) If sequences come back correct transform into expression strain ER2566 for pTWIN

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