Template:SBB-PCA

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(Design)
Current revision (16:01, 17 February 2012) (view source)
(Amplification)
 
Line 29: Line 29:
Recipe
Recipe
# 1 ul each outer oligo (10 uM)
# 1 ul each outer oligo (10 uM)
 +
# 1 ul purified pca product
# .5 ul phusion
# .5 ul phusion
# 10 ul 5x phusion buffer
# 10 ul 5x phusion buffer

Current revision

Gene Synthesis from Oligos (PCA method)

The general idea is to synthesize 500 to 1000 bp chunks of the gene of interest, and then clone those together to make the entire gene.

Design

  1. Use GeneDesign program (http://baderlab.bme.jhu.edu/gd/) to remove restriction sites. You should remove all relevant restriction sites in your product including AcuI, BseRI, BamHI, BglII, XhoI, EcoRI, PstI, XbaI, SpeI, NotI, AlwNI, BglI, AgeI, NgoMIV
  2. Put BglBrick ends on your sequence
  3. Use GeneDesign 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. Put your oligos in an excel file analogous to our oligo log google doc. There is a program on the Biomek to add the water and combine the oligos into tubes for you, so in then end you'll be working with 3 tubes of liquid: your pooled PCA oligos, a forward primer, and a reverse primer.

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

  1. 1 ul each outer oligo (10 uM)
  2. 1 ul purified pca product
  3. .5 ul phusion
  4. 10 ul 5x phusion buffer
  5. 5 ul 2mM dNTPs
  6. 32.5 ul H2O

Program

  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

  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

Program

  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. You should grow up, mini, and map 4 clones for gene synthesis, as we'll likely need to sequence more clones than we would for PCR-based part fabrication
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