Smolke:Protocols/Gene assembly

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{{Smolke_Top}}
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==DNAworks==
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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.
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Rough protocol:
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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. 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.
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The synthesis works by a two-step PCR process:
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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)
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An example assembly PCR thermal cycle:
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Step 1: 94C for 5 min
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Step 2: 94C for 15 sec
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Step 3: 62C for 30 sec (rule of thumb oligo melt -1C)
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Step 4: 72C for 50 sec (~1min per kb, although Roche Expand is a bit faster, read Expand instructions)
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Go To #2 for 29 cycles (30 cycles total)
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Step 5: 72C for 10 min
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Step 6: 15C forever (final hold)
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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).
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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.
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Troubleshooting:
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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
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If you have an especially difficult time troubleshooting this process, ordering full gene synthesis is probably a better way to get your gene.
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Reference:
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Hover and Lubkowski - http://www.ncbi.nlm.nih.gov/pubmed/12000848
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==Isis' version==
==Isis' version==

Revision as of 19:01, 17 February 2014

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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. 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: Hover and Lubkowski - http://www.ncbi.nlm.nih.gov/pubmed/12000848


Isis' version

Isis will fill this out soon...


Josh's version

Save yourself the trouble and get it synthesized.

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