Smolke:Protocols/Gene assembly: Difference between revisions
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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. | 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: | '''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. | 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: | The synthesis works by a two-step PCR process: | ||
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An example assembly PCR thermal cycle: | An example assembly PCR thermal cycle: | ||
Step 1: 94C for 5 min | *Step 1: 94C for 5 min | ||
Step 2: 94C for 15 sec | *Step 2: 94C for 15 sec | ||
Step 3: 62C for 30 sec (rule of thumb oligo melt -1C) | *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) | *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) | *Go To #2 for 29 cycles (30 cycles total) | ||
Step 5: 72C for 10 min | *Step 5: 72C for 10 min | ||
Step 6: 15C forever (final hold) | *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). | 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. | 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: | '''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 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 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. | *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' 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:
Isis' versionIsis will fill this out soon...
Josh's versionSave yourself the trouble and get it synthesized. |