Lidstrom:Sequencing with GeneWiz

From OpenWetWare

(Difference between revisions)
Jump to: navigation, search
(No priming: lots of "N"s in sequencing result)
(When Sequencing Fails)
Line 40: Line 40:
*dye blobs [[image:dye_blob_GeneWiz.png|thumb]]
*dye blobs [[image:dye_blob_GeneWiz.png|thumb]]
**too many terminating nucleotides remain after their purification -- see image on right.  A big peak covers the sequence you want.  They generally have it at the beginning, so you should start your priming ~ 100 bp away from the sequence of interest.  They happen on PCR products and plasmid & are best visually edited (ignored.)   
**too many terminating nucleotides remain after their purification -- see image on right.  A big peak covers the sequence you want.  They generally have it at the beginning, so you should start your priming ~ 100 bp away from the sequence of interest.  They happen on PCR products and plasmid & are best visually edited (ignored.)   
 +
 +
===[https://clims3.genewiz.com/Customer/Support/Troubleshooting/High%20Background.pdf High Background]===
 +
* what is the difference between High Background & non-specific?
 +
** Customer support says: the degree to which the background sequence
 +
** you can trust a sequence that fails because of high background more than you can non-specific
=== When it fails, try again! ===
=== When it fails, try again! ===
Line 48: Line 53:
**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.  
**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.
**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.
-
 
-
===[https://clims3.genewiz.com/Customer/Support/Troubleshooting/High%20Background.pdf High Background]===
 
-
* what is the difference between High Background & non-specific?
 
-
** Customer support says: the degree to which the background sequence
 
-
** you can trust a sequence that fails because of high background more than you can non-specific
 

Revision as of 12:18, 27 June 2012

Back to Protocols

Contents

Preparing Samples

  • 15 uL total including 5 uL of 5 uM primers.
    • Keep a fresh stock of 5 uM primers that you take good care of and replace often with fresh stock.
  • Red Flags:
    • Low concentration minipreps (<70 ng/uL) frequently fail when submitted to sequencing. It may be due to impurities that co-elute; if you submit many uL of this to meet the 500 ng/sample requirement, then you will have more of these impurities present. -Janet 3/20/2012

When Sequencing Fails

  • There are several ways it can fail & you can see the GeneWiz help for causes/solutions below. You can also watch this GeneWiz webinar. Don't hesitate to call GeneWiz customer support -- their customer service is incredible.
  • GeneWiz has very high standards for passing sequencing reactions. About 1/2 of your attempts may fail. Sometimes you can look at the trace file and decide it is good enough on your own despite a failed label.

Non-specific

    • multiple are present on your DNA or you accidentally have a mixture of DNA templates.
      • You can prepare a new miniprep, with the hope that your first one was contaminated with another plasmid. Otherwise, you have to assume the plasmid you built accidentally contains an extra priming site. Of course, miniprepping from single colonies will reduce the possibility of mixed templates.
      • Note: a plasmid can contain an extra priming site via pcr/assembly fluke OR the insert you add can contain a mispriming site.
    • hairpin present
    • The clone has multiple priming sites (can be on accident)

Poor quality

  • high background: there is a clear sequence but there is also a background sequence.
    • remnants of chromosomal DNA
    • primer degradation: recall that your sequencing is dependent on length. So if you have one population of primers that is 20 bp and one that is 18 bp, they will give two separate signals.
  • top heavy: beginning sequence is strong; peaks may even be cut off. As it gets longer, the signal dies off. This is due to improper primer:template ratio; your primer may be "used up".

No priming:

  • lots of "N"s in sequencing result
    • too much, too little, or no DNA
    • no primer or sub-optimal primer concentration
    • primer site not complementary
    • carry-over inhibitors in the reaction such as phenol, ethanol, EDTA, or salts

Early termination

  • hairpin present, repetitive sequence, or high GC content can terminate the rxn sequencing. Note: they have several "difficult sequencing" protocols that can be used; select them in the form.
  • too much DNA
    • results in smears. They can dilute the sample you sent and re-run it. Use 10ng/uL/kb of DNA.
  • bubble in capillary
    • can appear as a smear. You won't know when this happens, but customer support may be able to tell.
  • dye blobs
    • too many terminating nucleotides remain after their purification -- see image on right. A big peak covers the sequence you want. They generally have it at the beginning, so you should start your priming ~ 100 bp away from the sequence of interest. They happen on PCR products and plasmid & are best visually edited (ignored.)

High Background

  • what is the difference between High Background & non-specific?
    • Customer support says: the degree to which the background sequence
    • you can trust a sequence that fails because of high background more than you can non-specific

When it fails, try again!

  • 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, you should contact customer service and get approval. They will instruct you to put the old order number (for the ones that failed the 1st time) in the comments section on the new order you will submit with fresh template. Janet 3/20/12
    • If you need to sequence another 1/2 price rxn, you should probably chat with customer service and they will put a note on the account. They usually tell you to put the name of who you chatted with on the form, too.
  • 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.
Personal tools