Smolke:Protocols/Gene assembly: Difference between revisions
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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 | ||
Entered by Mike Siddiqui - February 2014 | |||
==Isis' version== | ==Isis' version== |
Revision as of 17:05, 17 February 2014
DNAworksHas 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:
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:
Reference:
Entered by Mike Siddiqui - February 2014 Isis' versionIsis will fill this out soon...
Josh's versionSave yourself the trouble and get it synthesized. |