Find restriction sites and fragment lenghts

note:
EMBOSS and ApE calculate fragment lengths differently;
e.g. EMBOSS gives sizes 3530 and 5505 for the Lambda x EcoRI/BamHI end fragments, while ApE shows 3531 and 5504.
This has to do with how the two programs deal with the position of the recognition site vs the position of the cut made by the restriction enzymes..

Use the program EMBOSS restrict. More...
Use the program ApE, a plasmid editor. More..

Number nucleotides in DNA sequences:

For example: use ApE to number the nucleotides in this sequence:

Use the Text Map feature

and if you would leave this default settings:

You would get this:

Alignment of two given sequences

For the alignment of two given sequences you can use the BLAST program bl2seq (Blast Two Sequences), for example to find out where a primer exactly anneales on a particular template.

Go to http://blast.ncbi.nlm.nih.gov/Blast.cgi and choose the nucleotide blast program in the Basic BLAST section:

Choose the Align two or more sequences option:

And enter a Query Sequence (e.g. the smaller of the two Standard GeneTech primers),
and a Subject Sequence (e.g. the sequence of pGT4)

..like this:

Then, scrol down to click the BLAST button:

The resulting output screen shows the position in the pGT4 sequence, where homology with the primer is found:

So, in this example, the smaller (17 nucleotides long) Standard GeneTech primer has perfect homology (is identical to) with the pGT4 sequence from nucleotide position 1164 to 1180.

If you would do the same for the longer (30 nucleotides long) Standard GeneTech primer, you would get this:

It shows you, that the longer primer is identical to the pGT4 DNA sequence from position 1586 to 1557, which means it is in the reverse orientation ("it anneals to the complementary strand and points backwards").

Both primers has also some (partial) homology in other parts of the pGT4 sequence.

Finding primer binding sites using ApE

In this example, the ApE program is used to find the binding site on pGT4 DNA for primer ggaaacagctatgaccatgattacgaattc.

Open ApE and paste the pGT4 sequence in the sequence field (or open the pGT4.ape file, if made before..). Make sure that after pasting a plasmid seq into ApE, the linear/circular button is set to circular!!

Go to Edit>Find (or use Ctrl+F)

Paste the primer sequence

and check also find rev-com of string (the primer may be identical to (a part of) the opposite pGT4 DNA strand..)

and click the Find Next button to get this:

As seen by eye, the sequence in blue appears to be the reverse-complement of the primer sequence.
The bar below the icons shows, that it's length is 30 nucleotides, and the binding site is from 1557 to 1586 on the pGT4 DNA.
The primer "points backward" since the 5' end is at 1586. Elongation of the primer in a PCR reaction will be from 1557 towards lower number postions ("backwards") on pGT4.

Tm of PCR primers

For an accurate calculation of the Tm of PCR primers you could use the Oligonucleotide Properties Calculator at http://biotools.nubic.northwestern.edu/OligoCalc.html/.

about ORF Finder

The ORF Finder (Open Reading Frame Finder) tool finds all open reading frames in a given sequence.
If you for example would paste the sequence of vector pGT4 (can be found on the Sequences page) in the paste-sequence field:

and click the OrfFind button (top left above the field) you would get this:

where ORFs are shown by green bars. The longest one runs from nucleotide position 284 to 1474. It's in the -1 (minus one) frame, so it starts at 1474 and stops after 284. This is the tetracyclin resistance gene of pGT4. If you click on that green bar (or the green square before 284), you get more detailed info about that reading frame:

[optinal] Try this:

Paste the pGT4 sequence in the or sequence in FASTA format field
Find the BamHI site ggatcc in the sequence in the field, using Ctrl-F
Change that into ggatcgatcc (the filled-in and religated BamHI site) giving the sequence of pGT4DB
Click the OrfFind button
Find out what happened with the tetracyclin resistance gene of pGT4 when it was "mutated" to pGT4DB (check the length of the -1 reading frame that starts at 1478...)