Lidstrom:Sequencing with GeneWiz

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**Always have good labels in your FASTA files to make sure you don't mix things up.  
**Always have good labels in your FASTA files to make sure you don't mix things up.  
**If you are making variants of a construct, you can compare your sequencing results to all of the things you are making.  First make a text document with each of the reference sequences you are creating by pasting them back to back in a text editor.  Include descriptions for each e.g. "> SH3-FLS in pSB1A3".  Paste your sequence from GeneWiz into the top box and all of the other sequences in the bottom box.  When the results are returned, click the "max score" button & it will put the match with the highest score first.  This is most likely your product, but a sequencing read can match to multiple designs depending on your project.
**If you are making variants of a construct, you can compare your sequencing results to all of the things you are making.  First make a text document with each of the reference sequences you are creating by pasting them back to back in a text editor.  Include descriptions for each e.g. "> SH3-FLS in pSB1A3".  Paste your sequence from GeneWiz into the top box and all of the other sequences in the bottom box.  When the results are returned, click the "max score" button & it will put the match with the highest score first.  This is most likely your product, but a sequencing read can match to multiple designs depending on your project.
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==When Sequencing Fails==
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*There are several ways it can fail.  You can submit 2 samples per order for a free repeat, but they must be on the same templates you submitted.
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*You can resequence as many as you like for 1/2 price and you have the choice to use new preps.  This allows you to use a fresh miniprep or primer batch.  If you want to submit fresh samples for 1/2 price, put in a new order and note which are free repeats, and their respective order number in the "Notes" section of the form.
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*Whether or not your repeat will be successful depends on several variables such as:
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**the concentration and quality of the sample -- if a "no priming" or "poor quality" samples is "no priming" or "poor quality" after you sequence again, the quality and quantity might be too bad to work the 2nd time.
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**you also may have a GC-rich region, hairpin or general difficult template that is causing binding issues, a problem with the binding site, a problem with the primer, etc.

Revision as of 12:45, 22 February 2012

Return to Protocols

Submitting Samples

  • Using the correct template mass and primer concentrations is crucial. If you submit less than the recommended ng of DNA, sequencing fails more often.
  • If you have a low concentration plasmid prep, you can increase the mass you send by sending 0.5 uL of 100 uM primers (the stock concentration) instead of 5 uL of 5 uM primers.

Understanding Results

  • Janet recommends doing a multiple alignment in NCBI's Blast webpage. Do not use the APE program's aligner, especially if you have a repetitive sequence.
    • If you get an imperfect match or fail to match to your expected sequence, you can loosen the stringency requirements by selecting "somewhat dissimilar" or "more dissimilar" instead of "highly similar."
    • Always have good labels in your FASTA files to make sure you don't mix things up.
    • If you are making variants of a construct, you can compare your sequencing results to all of the things you are making. First make a text document with each of the reference sequences you are creating by pasting them back to back in a text editor. Include descriptions for each e.g. "> SH3-FLS in pSB1A3". Paste your sequence from GeneWiz into the top box and all of the other sequences in the bottom box. When the results are returned, click the "max score" button & it will put the match with the highest score first. This is most likely your product, but a sequencing read can match to multiple designs depending on your project.

When Sequencing Fails

  • There are several ways it can fail. You can submit 2 samples per order for a free repeat, but they must be on the same templates you submitted.
  • You can resequence as many as you like for 1/2 price and you have the choice to use new preps. This allows you to use a fresh miniprep or primer batch. If you want to submit fresh samples for 1/2 price, put in a new order and note which are free repeats, and their respective order number in the "Notes" section of the form.
  • Whether or not your repeat will be successful depends on several variables such as:
    • the concentration and quality of the sample -- if a "no priming" or "poor quality" samples is "no priming" or "poor quality" after you sequence again, the quality and quantity might be too bad to work the 2nd time.
    • you also may have a GC-rich region, hairpin or general difficult template that is causing binding issues, a problem with the binding site, a problem with the primer, etc.
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