2. (2) On a computer paste or type in your coding DNA strand in the box at http:

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2. (2) On a computer paste or type in your coding DNA strand in the box at http://web.expasy.org/translate/ and clic on TRANSLATE SEQUENCE. Give the single letter aa sequence of any open reading frame(s) (those not having a STOP; 0 to 6 possible):

____________________________________ ____________________________________

____________________________________ ____________________________________

____________________________________ ____________________________________

3. (2) BLAST the coding DNA sequence again, this time scroll down thru Graphic Summary and Descriptions to Alignments and look in the results for an mRNA (predicted mRNA or protein) with the most similarity and the Sbjct numbers increasing from left to right clic on it. Note here the position numbers within the Sbjct DNA.

_______________________ _____________________

4. (3) Under that protein name there is an alphanumeric Sequence ID number, clic on it. That should give you a large readout of the protein primary structure containing your name or most of it and the associated DNA listed 10 bases at time. Find your name, count the amino acids from the N-terminus and multiply by 3, that is the position where your name starts in the DNA. Now clic and drag through 300 letters that encompass your sequence (e.g. if my first Sbjct number was 3524, I might select 3424-3724) and copy those highlighted bases. It is OK that the line numbers are included for now. Paste this into a Word document for easy access. Go to the primer designing tool at http://www.ncbi.nlm.nih.gov/tools/primer-blast/ and paste the 300 letters in the box. You may take a moment to look at the adjustable parameters but eventually skip down to the bottom and clic the Get Primers button. You would do this to amplify your name in PCR. Choose a primer set that encompasses you name. The optimal Tm difference between primers is less than 2degrees, what are your primer set Tm’s?

Forward ________________________ Reverse ________________________

5.(3)Copy the 300 base sequence from Word and then go to http://www.restrictionmapper.org/ and paste the sequence in the box. Clic on Map Sites and you will get a screen of non-cutters and cutters with the position of the cut in your DNA. If we wanted to use pQE -30 as a cloning vector, circle those of the following restriction enzymes that are present in your name 300mer that are also in the pQE -30 multiple cloning site?

StuI BamHI SphI SacI Kpn1

SmaI XmaI SalI PstI HindIII

If none of these cut, list the two restriction enzymes that would give you the largest fragment ____________________________________________________________________ If you are curious, there are a couple ways to approach this problem: you could add the restriction enzyme sticky ends at the 5’ of your primers and amplify up what would look like a restriction digested fragment. Alternatively you could expand your sequence from the 300ish to a much larger amplimer that would more likely include some of the listed enzymes.

6. (2) Note the first cutter on your mapperlist and use the back button to go back to the Mapper screen. Clic on that enzyme in the Select Individual Enzymes list to highlite and then clic on Virtual Digest. What is the size of the largest fragment?

NB These are some of the computer tools that I actually use in my research when I want to create some DNA to clone into a bacterium and eventually move a sequence to an expression vector like pQE-30 and have bacteria make my protein.

Explanation / Answer

2.i) M P G Y R L W L

ii) M A V A L E S

iii) M C S A R V V A C

iv) M I G R Q L

v) M S V G S C S

vi)  M A A V A S

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