Link to the LSC Nucleic Acid Facility.

Two recent sequencing attempts by the Nucleic Acid Facility have revealed problems that some of you might encounter.  The first example illustrates how an inverted repeat abruptly terminates the transcription reaction.  Note how the sequence ends around nucleotide 285.  The next 52 nucleotides are composed of an inverted repeat which probably snaps into a hairpin during the sequencing reactions (the sequence is: tcgagaagggttcaggcgtgggcgtcgacgcccacgcctgaacccttctcga).  I'm not aware of a simple solution to this problem.


The next example illustrates how G and C-streches shorten the length of readable sequence.   Presumably, GC base pairing is inhibiting the polymerase.  Typically, the sequences are strong for 500 to 700 nucleotides.  This sequence run has ended around 300 nucleotides.  Note the numerous stretches of C's between 250 and 280.  It may be possible to improve the sequencing reaction by running it at a higher temperature.  I recommend discussing this with Deb Grove if you notice that your sequences has numerous stretches of C's or G's.


(Update 5/10/03) If your  sequencing reaction fails, consider the possibility that the DNA is contaminated.  The Nucleic Acid Facility recommends PEG precipitating DNA. They can provide a procedure or one can be found here.  I used to recommend spermine precipitating the DNA.  However, a barage of poor results has lead me to suspect that residual spermine might inhibit the sequencing reactions. Both procedures should rid the DNA of small pieces of contaminating RNA as well as other contaminants.  The advantage of spermine precipitation is that it works well on concentrations of DNA below 100 ug/ml whereas PEG precipitation should be limited to concentrations of DNA above 100 ug/ml.

It is also prudent to make certain that you have selected an appropriate primer that anneals to your template.  I've found a local alignment program to be very useful.  Enter the sequence of your primer into the top window and the predicted sequence of your DNA into the lower window.  Run the program and look for a match in the output.  If there is not match, go backwards in your browser to the bottom sequence window, select reverse, and run the program again.

The PSU Nucleic Acid Facility has a number of sequencing primers that they will supply at no charge.
 
T7 TAATACGACTCACTATAGGG
T7T GCTAGTTATTGCTCAGCGG
T3 ATTAACCCTCACTAAAGGGA
SP6 CGATTTAGGTGACACTATAG
M13 Universal TGTAAAACGACGGCCAGT
M13-40 GTTTTCCCAGTCACGAC
M13-47 CGCCAGGGTTTTCCCAGTC
M13Rev CAGGAAACAGCTATGACC
M13R-48 AGCGGATAACAATTTCACA
Lambda GT10 AGCAAGTTCAGCCTGGTT
L GT10Rev CTTATGAGTATTTCTTCCA
LGT11 GTTGGCGACGACTCCTGGAGCC
LGT11Rev GACACCAGACCAACTGGTAATG

To possibly assist in finding alignments, paste the following into the top window of the local alignment program.
TAATACGACTCACTATAGGG
GCTAGTTATTGCTCAGCGG
ATTAACCCTCACTAAAGGGA
CGATTTAGGTGACACTATAG
TGTAAAACGACGGCCAGT
GTTTTCCCAGTCACGAC
CGCCAGGGTTTTCCCAGTC
CAGGAAACAGCTATGACC
AGCGGATAACAATTTCACA
AGCAAGTTCAGCCTGGTT
CTTATGAGTATTTCTTCCA
GTTGGCGACGACTCCTGGAGCC
GACACCAGACCAACTGGTAATG

Paste your DNA of interest in the lower window and run the alignment.  Examine the output for matches.  It helps to determine the location of landmark restriction sites so you can identify primers that will associate with the plasmid adjacent to the region to be sequenced.  The local alignment program only checks one strand at a time - you have to select reverse to check the opposite strand.  I find this difficult because the numbering of the nucleotides no longer matches the numbering of the sequence in DNA strider.  Therefore, I prefer to temporarily generate an anti-parallel sequence in DNA strider, enter this into the local alignment program and compare the output to a graphic of landmark restriction sites I've selected in DNA strider based on the anti-parallel sequence.