Sequence starts too far down stream

[Jason]

I have been having issues with the start of my sequences. The best example I can give is a plate that had three pGEM controls. The same amount of sample was loaded with the same amount of primer for all three controls, but all three had different sequence start sites. The first control started 28 bp downstream of the primer, the second started 87 bp downstream, while the third started 308 bp downstream. Does anybody have any idea why this occurs and what the best way to alleviate it?
Thanks.

[Daniel]

Hi Jason

Have you looked for dye blobs? The other cause of late starts is salt. Have a look at the raw data channels and see if there is a big difference to where the signal starts?

Daniel Tillett
Nucleics Support

[Jason]

It seems there was a small crack in the lid to the bottle we store our absolute EtOH. The lower than thought concentration was not ppt the smaller fragments. Thank you nucleics.

[Daniel]

Ah the ethanol absorbing water from the air problem :). Yes this will cause problems. I recommend that you use 95% ethanol from small aliquots to avoid this problem.

Daniel Tillett
Nucleics Support

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