In one of the region, the sequencing was not clear. Could I request Bio Basic Inc. to sequence again?

We would need you to first clearly tell us at which position is unclear to you and then we would go case by case.

I lost all data, could you send me another copy?

Yes. However, please note, all sequencing results are kept for a period of 6 months. Samples are kept at Bio Basic Inc. for 1-2months.

I have a 4kb PCR fragments, can Bio Basic Inc. sequence through the full length?

For PCR >2kb, we recommend to first subclone into a vector. The insert is more stable in a vector and sequencing results will be easier to achieve.

I have a mixed base pair in a position, how come I do not see the mixed base in the report?

When preparing the report, Bio Basic Inc. assumes each sequencing sample is from a pure single population rather than mixed. Based on this assumption, the higher/stronger peak is being reported and lower/weaker peak being treated as background. If your sample contains a mixed population, please kindly notify us.

The bands are present and strong, but irregularly spaced, or with mixed colors. The technician may have reported "superimposed sequences" or used the phrase "peaks on peaks"?

If you see this, you usually have two sequences superimposed on each other. There are several common causes:

• 1. The sequencing primer binds to two (or more) sites on the template.
• 2. There are two (or more) templates present.
• 3. This was a PCR reaction, and you didn't remove the original primers.
• 4. This was a PCR reaction, and one primer generated *both* ends.
• 5. This was a PCR reaction, and there is more than one amplified species present.

Your sequence proceeds normally, then the bands abruptly become much smaller.

Secondary structure in the template is the most likely cause of this problem. The polymerase is presumably unable to progress through some stem-loop form. A few possible solutions:

1. 1. Try resequencing by selecting 'siRNA construct' as your DNA type.
2. 2. Try to sequence from another primer at a different position (closer or further).
3. 3. Sequence the other strand.

We maybe able to use special cycling conditions and/or special reagents that help the polymerase to push through this region. We cannot do this routinely, as it involves extensive optimizations.

Your sequence proceeds normally, then the bands abruptly vanish.

This usually happens when the template DNA has simply stopped, for example if it was restricted at a downstream site or if the template was a PCR product. This may also be caused by an extremely stable secondary structure.

The sequence looks great until it hits a polyA (or polyT), and then the bands rise and fall in waves.

This is called "polymerase slip". It happens when the growing strand temporarily dissociates from the template, then reassociates at a different spot - say, one nucleotide forward or back from where it started. If this happens often enough (as it will on polyA or polyT templates), every individual band becomes a family of closely-spaced peaks giving a 'roller coaster' look to the chromatogram. Try sequencing in the other direction from the opposite strand, or try another primer either closer or further from the homopolymer region.

You offer 24hr to 48hr service, but it has been 2 days and I still have not received data?

24hr to 48hr is AFTER we receive the samples at Bio Basic Inc. If you have more than 5 plates, longer turnaround is expected.