"Clone" software is a brand of windows software that allows the importing of genetic (i.e. phage genomic) data, and the analysis of this data for cutting with restriction enzymes.  This software allows a scientist to "understand the DNA molecule he or she is building."  It also allowed us to predict the Restriction Fragment Length Polymorphisms patterns we expected to see after restriction enzyme digestion of phage DNA.  Other brands of software are available, if desired.  The clone software is available at http://www.scied.com/sescat.htm (Clone Manger 7, i.e.).  **The company does offer a free demo disk.  The program is really easy to use. 

This program gives specific values within DNA sequence data, where restriction sites are.  In addition, the software generates a genetic map of where the sites are within a genome.  Shown here are the Cla 1 sites within the Bxb1 mycobacteriophage genome, in a map generated by the Clone software: 

Complete Cla1 digestion would cut the DNA at 8228, 8737, 11031, 12704, 20352, 24070, 26285, 29745, ....etc unto the last cut at 50,081 base pairs (bp).  Each piece of DNA flanked by Cla1 sites would be a DNA band that migrates separately during electrophoresis. 

(An advanced assignment, if using the D29 phage in your teaching or studies, is to have students predict the size of DNA fragments from the pubmed sequence data.  My high-school students were able to do this with very little of my own help, using the methods herein.) :

How to use Clone to predict enzyme cuts on a phage: 

 

1. Go to www.pubmed.com and in the tab next to the search bar; switch from PubMed to genome, then type in the name of the Mycobacteriophage in the search bar.
2. From the results that are displayed, choose the sequence you which to analyze.
3. On the data page, scroll to the bottom of the, and look for the line that says “COMMENT   PROVISIONAL REFSEQ: This record has not yet been subject to final NCBI review. The reference sequence was derived from” Then click the link after it. I.e., “The reference sequence was derived from AF022214.”
4. Scroll down to about 3/5^th of the way down the page, and look for the line that says ORIGIN, followed by combinations of A’s, T’s, G’s, and C’s. Highlight 1 to the bottom of the page, and press Ctrl + C to copy the data. Note: do not select anything after and including the // on the bottom of the page.
5. Open SECentral (Clone) in C:\SECentral.
6. Type Ctrl + N to open a new document within the program, and select paste sequence, the click finish, check Do not Auto Scan, the OK. (Phage genomes can be thought of as linear sequence, therefore leave as linear.)
7. SAVE YOUR SEQUENCE NOW, BEFORE YOU WORK WITH IT, OTHERWISE YOU WILL HAVE TO REPEAT THE ABOVE PROCEDURE.
8. Click on RMap on the bottom of the front box, and find the enzyme you want to map with.
9. Right-Click on the name of the enzyme you are going to map, and left-click enter sites to map.
10. Click on Map on the bottom of the front box, and you will see the where the enzyme appears in the sequence.

USING CLONE TO COMPARE DIFFERENT PHAGE RESTRICTION DIGESTION FRAGMENTS (SIZE VRS. SIZE): Some things that can be done with the software.  

 

1.) Every non-matching base pair can be shown when juxtaposing an infinite number of genomes. Use: Compare Two sequences, Binoculars over yellow paper.

Photoshop Instructions for coloring/making invisible

1.)Create a Ellipse using the Ellipse tool.

2.)Go to the Layer under the menu, then select layer style, then click Blending Options, Once this box opens, select opacity.

3.) The lower the opacity, the more transparent the circle becomes, visa-versa.