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C's after T's are usually small
If there is more than one G after an A, the first G is small.
If there is more than one C after a G, the first C is small.
Sometimes in a string of 4 G's , the 2nd or 3rd G is small.
T's after G's are usually small.
In a string of 4 or more A's, the second A is usually small.
Why did my sequencing fail?
-Poor quality template
If you have not purified your DNA properly (we recommend Qiagen) or if there are contaminants such as ethanol, salt, PEG, protein or genomic DNA, you will get very messy results or no sequence at all.
-Unquantitated or incorrectly quantitated DNA
Your results from automated DNA sequencing are very dependent on the amount of DNA that we use in the reaction. If you don't quantitate the DNA properly and we don't use enough, you may not get any signal, which means no results. Also keep in mind there is also a problem if too much DNA is used. Please take an O.D.- 260/280 which should be between 1.7 and 1.9. A smaller ratio would indicate the prescencee of protein and a ratio higher than 1.9 would indicate the prescence of RNA. This can be fixed by an RNAse treatment.
-Samples in TE
Your samples should always be resuspended in ddH2O rather than TE buffer. EDTA chelates magnesium and Taq needs magnesium in the reaction for it to work.
-Primers
The primers should be at least 18 bases long with a Tm between 55 and 65c. They should be binding 100% with the target DNA and have no hairpins or long repeats (more than 3 of the same base in a row). Please remember, even if a primer works for PCR it doesn't necessarily mean it will work with automated sequencing.
-Host strain
The host strain used for specific template preparation can impact the template quality. HB101 and DH5alpha work well, MV1190 and XL1 Blue show variability in results and JM101 usually yields poor quality DNA for sequencing.
-High GC content, secondary structures, long repeats and long homopolymer regions
If you know your template has any of these, please make note of it on your order form so we can run them accordingly. We do have a couple tricks up our sleeve to deal with these problems. Unfortunately, they don't always work.
Suggestions?
paul_morrison@dfci.harvard.edu How the site works.