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Center for Protein and Nucleic Acid Research

DNA Data Comments and FAQs

What do the comments mean?

1. "blank" - Blank means that there was no priming and extension. Below are listed the possible reasons and remedies for blank samples.

a) Sample contains inhibitors, such as too much salt, EDTA, certain dyes, heme compounds. Remedy: Apply sample to Amicon Centricon-100 columns (Note for smaller DNA fragments you may need other columns with a lower MW cutoff) and follow our DNA Clean-up procedure. Some sample columns are available for you to try out. They are located in our office on the shelf above the computer at MB101.

b) Sample DNA template is degraded. Remedy: Run Agarose gel and check yield by A260.

c) Inaccurate quantitation. Quantitate sample in dH2O only. EDTA and other compounds can throw off the quantitation.

d) Primer choice not optimal or primer is degraded. Have you used the primer before? Is it really old? Too many freeze-thaw cycles will degrade primers.

e) Tm of primer is too low. Your primer should have a calculated Tm around 58-62 C.

f) Lastly, excessive supercoiling may be the culprit. Try submitting the PCR product or linearizing it with restriction digestion.

Our "blank" sample policy: If your entire set is blank, one sample will have the comment "blank rr " on it. The "rr" means that we will take the remaining portion of your sample and react it at no charge. If the rr is labeled "blank 2x" that means that the sample was blank again. If the rr is labeled "too light 2x ", that means that the primer is working but that the signal was too light to get a full read. This may indicate a salty sample or you did not have enough DNA template in the sample. If the sample was part of a set, the remainder will not be reacted a second time. You must clean the samples up and resubmit them at your cost.

2. "too light" - Too light means that the G-signal was too low and to get off a full read (whatever read length you requested). This can be caused by too little DNA, primer, or poor primer binding, although it is usually caused by low quality DNA. Try desalting the sample as discussed above in the blank samples section.

3. "mixed" - Mixed means that there was more than one signal. This is usually caused by multiple priming sites or multiple primers in the sample.

4. "clean then mixed" - This means that there is a mixed colony or multiple templates. The mixture usually starts at the cloning site and consists of one clone with an insert and one without the insert.

5. "falls off" - This may mean that the sample was too salty or there was too much template in the sample due to inaccurate quantitation. Try our DNA cleanup procedure mentioned above and quantitate the sample in dH2O only.

6. "short" - This could be a PCR product or there are strong secondary structures or hairpin loops that cause the enzyme to fall off abruptly and stop extending. This can sometimes be overcome by adding DMSO. Resubmit your sample and check "GC-rich".

7. "pr pk shp" - This means that a majority of the peaks on the electropherogram had a poor peak shape, enough so, that the peaks were unable to be called by the software. This may be a result of too much DNA template or protein contamination in the sample.

8. "rough spot" - This means there is a rough spot in the sequence, such as a run of one nucleotide or a poly TA region, and the read following such region has problems.

9. "rl" - This means that we are going to reload this sample again to see if the original poor result was due to some problem that may have ocurred on the instrument on that particular day. The extension rl will be added to sample name. A sample that has been reloaded, and the result is no different from the first will say "no better" or "same" in the comment section.

Each time a sample is resubmitted, normal charges will apply.

Thermal cycling conditions are, 96C denaturation, 50C anneal, 60C extension.