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Author Topic:   Sequencing Trouble
Shelley
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posted 07-08-2005 04:18 PM     Click Here to See the Profile for Shelley   Click Here to Email Shelley     Edit/Delete Message Reply w/Quote
Could anyone help me solve a sequencing problem? I am developing an assay but one fragment is being very stubborn. Using Big Dye v.1.1, the reverse segment turns out beautifully on the ABI 3100, but the forward seqment has very low intensity and very high background. I've tried using more DNA template and altering the thermocycling to allow for annealing at a lower temperature, but to no avail. The fragment is not GC rich, and under the same conditions all the other fragments work fine. Has anyone encountered a similar problem? Any suggestions?

IP: 142.20.131.73

williamssp
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posted 07-13-2005 09:05 PM     Click Here to See the Profile for williamssp   Click Here to Email williamssp     Edit/Delete Message Reply w/Quote
Shelley,

Has your forward primer been validated on another sample? If so, has it been freeze/thawed many times and possibly degraded? You might try designing another forward primer or two. If you know the forward primer is good, secondary structure could be causing problems on the forward strand. You might try linearizing or digesting your sample to smaller fragments and resequencng.

Good Luck!

IP: 12.18.36.40

Shelley
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posted 07-15-2005 02:10 PM     Click Here to See the Profile for Shelley   Click Here to Email Shelley     Edit/Delete Message Reply w/Quote
Thank-you for the advice! The primer I have been using has not yet been validated, so I will try next some different forward primers.

IP: 142.20.131.73

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