CGG repeat sequencing

[Anonymous]

I am trying to sequence the FMR1 gene. My first PCR is OK but I have a problem to get a satisfactory result in the second PCR. Does anyone knows a sequencing protocol that works with hight CGG repeats zone?
Thank you.

[Daniel]

Hi Alzenira

There are a couple of things you can do:

1. Incorporate 7-deaza-dGTP into the PCR you use the PCR amplify the FMR1 gene. This reduces the binding energy of the GC pairs and so makes it easier to sequence.

2. Add Betaine (~1M) to the sequencing reaction. This will help melt the

3. Use a mix of BigDye 3.1 and the dye rodamine chemistry. From memory the ration should be 1:4.

Daniel Tillett
Nucleics Support

[Anonymous]

Muchísimas gracias. Me has ayudado mucho.

[Author]

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