Template:SBB-Protocols PCR4

Gene Synthesis from Oligos (PCA method)
The general idea is to synthesize ~500 bp chunks of the gene of interest, and then clone those together to make the entire gene.

Design
Use GeneDesign program (http://baderlab.bme.jhu.edu/gd/) to remove restriction sites You should remove all relavent restriction sites in your product including AcuI, BseRI, BamHI, BglII, XhoI, EcoRI, PstI, XbaI, SpeI, NotI, AlwNI, BglI
 * 1) Roughly every 500 bp, make sure there's a restriction site you can use to clone chunks together.  If you're lucky, you can break the gene at natural gly-ser pairs, and just use normal BBB assembly.  If you don't have gly-ser pairs, you have to introduce silent sites for later assembly.
 * 2) Put biobrick ends on each chunk.  So, if you had a two chunk gene that didn't have a gly-ser, which you were assembling at a HindIII site, it would look like:
 * 3) 5 leading-EcoRI-atg-BglII-FIRST_HALF-HindIII-BamHI-5 trailing
 * 4) 5 leading-EcoRI-atg-BglII-HindIII-SECOND_HALF-BamHI-5 trailing
 * 5) Use GeneDesign program (http://baderlab.bme.jhu.edu/gd/) to design your oligos Click the 50bp button--this will keep the lengths of your oligos under 60bp so that you can order on the lowest scale.  It will help to order your oligos in PCR plates and array your oligos in some meaningful way.  There is a program on the Biomek to add the water and combine the oligos into tubes for you.

Assembly
OK, so you've got a bunch of oligos, now what? First, use this recipe and program to do initial assembly of the oligos (do a separate one of these reactions for each chunk you're assembling): Recipe
 * 1) 38 uL ddH2O
 * 2) 5 ul 10x expand buffer
 * 3) 5 ul 2mM dNTPs
 * 4) 1 ul oligo mixture (100uM total, mixture of oligos after combination of 100uM stocks)
 * 5) 0.75 ul Expand polymerase

Program (can run JCA/PCA1)
 * 1) 2 min initial denature at 94oC
 * 2) 30 sec denature at 94oC
 * 3) 30 sec anneal at 55oC [This should be the overlap temp of your oligos - vary as needed]
 * 4) 30 sec extension at 72oC
 * 5) repeat 2-4 30 times total

Amplification
Now, you need to do an amplification of the correct full-length chunks. Clean up the assembly reaction with a zymo column; don't bother running it on a gel - it'll be a smeary mess and won't really help you. Save the purified product in case this step fails! For the amplification reaction, do a normal phusion program with 1 ul of the cleaned up assembly reaction as template, and using the outermost oligos for the chunk. That is: Recipe Program
 * 1) 1 ul each outer oligo (10 uM)
 * 2) .5 ul phusion
 * 3) 10 ul 5x phusion buffer
 * 4) 5 ul 2mM dNTPs
 * 5) 32.5 ul H2O
 * 1) 2 min initial denature at 94oC
 * 2) 30 sec denature at 94oC
 * 3) 30 sec anneal at 60oC [This should be high, as your outer oligos now have a huge overlap with the correct product]
 * 4) 30 sec extension at 68oC
 * 5) repeat 2-4 30 times total

Re-Assembly
In case the first amplification fails, you can re-amplify using the first amplification as a template to direct the oligos in the second round: Recipe Program
 * 1) 37 uL ddH2O
 * 2) 5 ul 10x expand buffer
 * 3) 5 ul 2mM dNTPs
 * 4) 1 ul oligo mixture (100uM total, mixture of oligos after combination of 100uM stocks)
 * 5) 1 uL of purified assembly reaction from previous round
 * 6) 0.75 ul Expand polymerase
 * 1) 2 min initial denature at 94oC
 * 2) 30 sec denature at 94oC
 * 3) 30 sec anneal at 55oC [This should be the overlap temp of your oligos - vary as needed]
 * 4) 30 sec extension at 72oC
 * 5) repeat 2-4 30 times total

Then re-do the amplification step. You can iterate these until you get a full-length product.

Cloning
And finally, you have to get everything in vectors and clone the chunks together.
 * 1) Clean up the amplification reactions with a zymo column
 * 2) Digest the product and desired backbone with Eco/Bam
 * 3) Run everything on a gel, and cut out appropriate bands
 * 4) Proceed with cloning as usual
 * 5) Verify the sequence of 2-3 clones for each chunk - so far, I have a 62.5% correct clone rate.
 * 6) Clone the chunks together to make your full length gene!