I tried to do the Sanger sequencing using big Dye terminator 3.1 and ABI genetic analyzer 3130xl machine.
I have got several results, but it was high peaks at the position around 60-80nt of every sequence results. Relevant photos have been enclosed.
Cycle sequencing thermal protocol is following by standard.
master mix:
Big dye 3.1 - 1ul
ddH2) - 5ul
Sequencing buffer - 2ul
primer 4.5pmol/ul - 1ul
template - 1ul
How to fix this problem?
What are reasons of these??
Please, give me some comments.
- How are you cleaning up your big dye sequencing reactions ? Ethanol precipitation or some sort of column like DyEx to remove spent terminators ?
- It has been my experience in the past that if excess terminators are present they can produce these over arching slightly amorphous secondary peaks called dye blobs
- Further if they are present in high excess or your big dye kit is old they can concatenation or form oligomer structures that obscure sequence further down the read. Your structure in that position look like monomers
- The best way to remove is column purification rather than simple ppt of sequencing reaction products
- Finally with older big dye kits in particular if you heat denature the sample prior to loading with your formamide loading dye this can lead to breakdown of big dye and the contaminating traces you see
- Thus:
Hi, you can find helpful information here:
https://www.nucleics.com/DNA_sequencing_support/DNA-sequencing-dye-blobs.html
http://cgrb.oregonstate.edu/sites/cgrb.oregonstate.edu/files/files/CoreLab/ab_sanger_troubleshooting_guide.pdf
Is it always the same PCR product and can you see it in all capillars?
Please check some previous runs (different PCR products). If you see the same picture in all capillars it could be problem in capillaries.
And sometimes, in this case helped me a simple thing.
During the elution of sequence product (after its cleaning) I pipet the sample up and down several times.
- How are you cleaning up your big dye sequencing reactions ? Ethanol precipitation or some sort of column like DyEx to remove spent terminators ?
- It has been my experience in the past that if excess terminators are present they can produce these over arching slightly amorphous secondary peaks called dye blobs
- Further if they are present in high excess or your big dye kit is old they can concatenation or form oligomer structures that obscure sequence further down the read. Your structure in that position look like monomers
- The best way to remove is column purification rather than simple ppt of sequencing reaction products
- Finally with older big dye kits in particular if you heat denature the sample prior to loading with your formamide loading dye this can lead to breakdown of big dye and the contaminating traces you see
- Thus:
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