Smolke:Protocols/Gene assembly

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(DNAworks)
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'''Reference:'''  
'''Reference:'''  
*Hover and Lubkowski - http://www.ncbi.nlm.nih.gov/pubmed/12000848
*Hover and Lubkowski - http://www.ncbi.nlm.nih.gov/pubmed/12000848
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Entered by Mike Siddiqui - February 2014
 
==Isis' version==
==Isis' version==

Current revision

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DNAworks

Has worked for Mike for various gene synthesis targets lengths ~300bp - 1500bp. More difficult for >1500bp. Use Josh's gene synthesis method or gBlocks and Gibson assembly for longer sequence genes.

Rough protocol:

Identify your target amino acid sequence. Plug it into DNAworks online software with your preferred excluded sequences (restriction sites, barcodes, screening primer sequences, etc.) and rough parameters - a rule of thumb that has worked in the past is 70 nt oligo length and ~63C annealing temp. You will get a set of primers out of the software.

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

PAN oligo synthesis works for ordering all of these primers with enough fidelity to get a working full synthesis. This set will be an even number of oligos 1 - n, oligo number #1 and #n are the full length amplification oligos.

The synthesis works by a two-step PCR process:

First run an assembly PCR with all the oligos excluding the full length amplification oligos #1 and #n. A quick recipe for this PCR is to mix 1ul from each of the assembly oligo stocks (diluted to 100mM) and use 5 ul of this mix as the input for the assembly PCR. Roche Expand polymerase has been the typical polymerase used in lab for the DNAworks PCR assembly. Run a small assembly reaction (25-50ul)

An example assembly PCR thermal cycle:

  • Step 1: 94C for 5 min
  • Step 2: 94C for 15 sec
  • Step 3: 62C for 30 sec (rule of thumb oligo melt -1C)
  • Step 4: 72C for 50 sec (~1min per kb, although Roche Expand is a bit faster, read Expand instructions)
  • Go To #2 for 29 cycles (30 cycles total)
  • Step 5: 72C for 10 min
  • Step 6: 15C forever (final hold)

Use 1-3 ul of the product from this reaction as the template DNA for the second PCR reaction, which is basically a normal (30 cycle) PCR amplification reaction where you use the full length amplification oligos (#1 and #n from the DNAworks output).

Run 1ul of each of these reactions out onto a gel to assess the success of the assembly. You should see a smear of DNA from ~100bp up to full length as the product of the first reaction and a tight single band at the expected length as the product of the second reaction.

Troubleshooting:

  • If you get smaller non-specific bands you can gel extract the band of the desired length and re-run a PCR with the end primers
  • If you still get a smear at the second step, sometimes you can run a third PCR with the end primers and the second PCR as the product and get the correct length band as the product
  • If you have an especially difficult time troubleshooting this process, ordering full gene synthesis is probably a better way to get your gene.

Reference:

Isis' version

Isis will fill this out soon...


Josh's version

Save yourself the trouble and get it synthesized.

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