The presence of inhibtors (i.e., salts, ethanol, phenol) can be introduced during the isolation of the plasmid. A common source can be the silica based commercial spin columns found in various kits from Qiagen, Promega, Zymo Research, and others. Binding of the DNA is carried out in the presence of an alcohol solution containing chaotropic salts. The wash step prior to the elution step can be problematic if all of the alcohol and associated salts are not removed. The geometry of the columns with the use of an angle rotor can leave residual binding buffer which in even small quantities can inhibit the sequencing reaction.

You can confirm that residual buffer remains by transferring the column to a new tube after the wash step and spin at the suggested g-force for an additional 5 minutes. You will likely observe an additional small volume. There are a couple of simple steps that will improve your signal intensities and reaction success:

1. Spin the column 3 minutes longer than the recommended time at the wash step to ensure removal of binding buffer.
2. After decanting the buffer, repeat the spin for 2 minutes to dry the filter.
3. Rotate the column 180 degrees to remove any buffer trapped on the retaining ring. (The Zymo Research kits don’t have a problem with trapped liquid on the retaining ring due to their design)

The Genomics Core currently uses Fisher Scientific (Cat. #05408129) 1.5ml MCT Graduated tubes for sample submissions. Our Tecan robotic system has been calibrated to the dimensions of this tube which restricts us from accepting any other tube. The dimensions of other tubes may result in improper volume transfer or damage to our robotic system. A bag of 500 tubes may be purchased at a discount price from the Genomics Core through the Enzyme Freezer Program.

The Genomics Core requests 16 ul to ensure consistent transfer volumes from the tube to the 96-well plate by our robotic liquid handler. The volume accounts for planned excess and dead volume. Less is requested with 96-well plate submissions since a transfer step is omitted as the researcher is submitting sample and primer already in the 96-well plate.

The 3730XL DNA sequencer uses a 96-well format. Plates are loaded in chronological order in which samples are submitted. Your order may be split between two plates if there are more samples than remaining wells in the plate. This can easily be confirmed by looking at the well position of your last sample with data. If marked as being loaded in G12, this indicates that your remaining samples will be the first loaded on the next 96-well plate. Data should be received no later than the next day.

There are situations in troubleshooting sequencing results when potentially noisy data must be distinguished from a failed reaction with only background noise. This scenario can only be sorted out by viewing the chromatogram. Sequence Scanner v1.0 is freeware provided by Applied Biosystems for viewing trace files. Open the file within the Sequence Scanner software. Select the Annotation tab at the bottom of the open chromatogram window. The "Average Raw Signal Intensity" will be >1000 units for sequence with strong signal. Signal intensities less than 100 will indicate background signal only.

Any sample can be deleted from an order. Begin by logging in to your dnaLIMs account. Select "View Your Requests". The next window will allow you to delete the sample by entering the requisition number and selecting "Delete Request". Click on the "Submit" tab and the sample has been removed from the order. The same can be done for an entire order.

Caution: Deleting an order removes all the samples in that order!

Adding 1.3 µl of DMSO to the 16 µl submission volume will result in a 5% DMSO concentration in the sequencing reaction.

The ends of pCR™II and pCR™2.1 vectors are palindromic sequences, since both ends must accommodate the TOPO binding sites. Therefore, there is an inherent potential for secondary structure once the vector is ligated. This does not usually affect sequencing or PCR results, as cycling conditions are usually sufficient to overcome potential secondary structures that might form. However, certain inserts might enhance the formation of such secondary structures by the regions flanking the cloning site. Strong secondary structures may inhibit the cycle sequencing reactions commonly used in automated sequencing. If you suspect this is a problem for your clone, here are a few things to try:

1. Increase the annealing temperature of the cycling, or even try a two-stage cycling reaction.
2. Add DMSO to the reaction. This may reduce secondary structure formation. However, beware that it may also affect the fluorescence or some other portion of the reaction.
3. Design primers internal to the insert.