Easy Cloner v 1.0

Easy Cloner is a point-and-shoot cloning simulator for the IBM PC. The

program was described in an article in 'Biotechniques' in June 1991, and

this file adds some further information to help you use it.

I. The program consists of one file called EC.EXE plus another called

ECICONS.

II. For practice the distribution disc includes files for the SV40

virus and plasmid pBR322. The files are called SV40.SEQ, SV40.MAP,

PBR322.SEQ and PBR322.MAP. To check the program out, try cutting the Pst

fragment out of SV40 and cloning it into the Pst site of pBR322. Then, on

the new map, label the SV40 part of the new plasmid and rotate or invert

it until it looks how you like it. Write comments into the comment part

of the sequence file, name the new plasmid and its disc file, print it out

and save it to the disc.

IIa. Here are some details on how to do all that. You can call up

the program with the SV40 map by typing EC SV40 at the C:> prompt.

Alternatively you can start up by typing EC and then use icon 2 to

load SV40. To find its first Pst site, use the eyeglasses icon, then

type CTGCAG on the top line. Press Enter and the current position

should be moved to the Pst site at base number 1988. To mark this

site click on the scissor icon. Find and mark the next Pst site (at

base 3204) in the same way. Use the save icon (icon 7) to save the

fragment in the file TESTTUBE.SEQ. You will be asked whether to use

'sticky ends'. Answer 'Y' each time. To move to the pBR322 file,

click on the disc icon and choose L (load). When it asks 'Abandon

changes in file' answer 'Y' (if you answer 'N' the SV40 file without

the Pst fragment will be saved and will replace the original SV40 file

on the disc. The old file will however be kept as a .BAK file). Find

the Pst site in pBR322 using the eyeglasses icon as before. It should

be at base 3609. Bring in the SV40 fragment that you saved by using

the insert icon (icon 8). It will have 'sticky ends' and the Pst site

will be recreated at each end of the insert, as you will see.

To give the new plasmid a new name, first rename the file, with the

disc icon. Then go to the label screen with the label icon (icon 9).

Re-label the plasmid there as well, and type in the lables you want

your new plasmid to have. For example you could add a 'gene' from

bases 3609 to 4680 which has a lable saying 'SV40'. Make it level 1,

2, or 3 so as to make it stand away from the plasmid circle. Press

Esc to get back to the main screen. Go to the comment screen with the

comment icon (the last icon), and delete anything you don't want. Add

comments to explain how you made the new plasmid. Press Esc to get

back to the main screen. Use the printer icon (icon 10) to get a hard

copy of your map. You will be given a choice on the top line of the

screen of either sending the map to a dot matrix printer, or saving it

in a plotter file. Use the disc icon to save the sequence and the map

data to the disc. The rotate icon (icon 3) and the mirror (icon 4)

will rotate or make a mirror image of the map and adjust the DNA

sequence at the same time.

III. The .seq file must be in the Stanford/Molgen format. This

means that comments in the file must be in lines that start with a

semi-colon. These lines are up to 79 letters in length. After the

comments come a line that does not start with a semi-colon - this is

usually the name of the sequence, but can be any other text or even

left blank. The sequence starts on the next line, and can be any

length. The sequence ends with a number 2, which shows that it is a

circular sequence.

IV. To incorporate a plasmid map into a Word Perfect document it must

be written out to a plotter file using the printer icon. If you use

the SV40 files, you will get a plotter file that is called SV40.HPG.

Now call up the program GRAPHCNV.EXE, which is supplied with Word

Perfect to convert the file to one called SV40.WPG. It can then be

incorporated directly into a Word Perfect document using the commands

Alt+F9, U, C, F, and typing SV40.WPG. If you have Draw Perfect,

which is sold by the Word Perfect corporation at a very high price,

you can modify the image before putting it into Word Perfect. Word

Perfect alone will limit you to a few editing options such as

rotating, enlarging or shrinking, and moving the map around the page.

V. The .SEQ and .MAP files are modified simultaneously while you use

Easy Cloner. Do not make any changes in the length of the .SEQ file

with some other program because that will get it out of synchronization

with the .MAP file. If you have to change the length of the sequence

in the .SEQ file then it would be best to erase the .MAP file

completely. It will be recreated next time you use Easy Cloner,

although you will have to add back all of the plasmid labels and so on.

VI. To edit a plasmid for which the DNA sequence is not known you can

create a .SEQ file with a word processor that contains the right number

of 'bases' - just use letter X or something like that.

VIII. To run Easy Cloner without a mouse, use the keypad keys to move

through the sequence. The left and right arrow keys move one base at a

time and the other keys go in bigger jumps. The F1 key selects icon

1, and so on.

IX. The label-editing screen is for adding features to the plasmid

map. Sites or genes can have 4 levels. Level 0 is at the level of the

circle itself, and level 3 is the furthest out. Genes are defined by

two base numbers, the first being lower unless the gene crosses base 1.

'Direction' must be '+' to make a clockwise arrow, '-' to make a counter

clockwise arrow, or be left blank to make a line with no arrow head.

The base numbers are automatically updated when the plasmid is rotated

or reversed, or if bases are inserted or deleted. 'Label' is the text

that marks the plasmid site or gene. 'Plasmid labels' are what is shown

in the center of the plasmid and are not updated automatically. Site

labels, Gene labels and the second Plasmid label can start with the

symbol '#' in which case the map will show the actual base number and

will be updated if the plasmid is modified.

X. To adjust the position of a label put the mouse cursor on it - the

shape will change to a cross. Press the left mouse button and hold it

down while you drag the label to its new position. When the map is

printed the postions of the labels might not seem quite right, due to

the different text heights with some plotters and printers. In that

case you might have to move the screen labels to a position that does

not look so good on the screen, but does look right on the hard copy.

XI. To clone in a fragment from the disc in the reverse

orientation, simply make the map into its mirror image, clone in the

fragment, than make the mirror image of the whole new plasmid. You can

not reverse the saved fragment on the disc, but this method gives the

same result.

XII. You might need to splice in a file that was produced by some other

computer program. This can be done just as long as the file is named

TESTTUBE.SEQ. It must not have any comments or punctuation, but can

include carriage returns or base numbers. There will be no conversion

between lower and upper case letters, nor any check as to which letters

of the alphabet are used.

XIII. Easy Cloner tries to detect what kind of monitor you are using, and

then sets the video mode as best it can. You can force Easy Cloner to run

in different video modes by appending a letter to the command line. For

example to edit the SV40 files in CGA mode you type EC SV40 C. To run in

EGA mode you type EC SV40 E. For VGA mode use letter V, for Hercules mode

use H and for monochrome EGA mode use M. Obviously this will only work if

your computer has the hardware to support the mode you choose. There are

two reasons for which you might want to change the video mode. Firstly, on

a PC with a small VGA monitor the EGA mode might be easier to read than VGA,

or your PC-compatible might have a screen that looks better in some modes

than others. Secondly, you might wish to capture a screen image for later

editing by a graphics editor, and the capture might work best with a

monochrome image. An example is the FRIEZE program supplied with

Microsoft Paintbrush. However the resolution of the image will then be

limited to how many dots your monitor shows; the best resolution for a

printed plasmid map comes from using a plotter or a transfer to a plotter

file.

XIV. I wrote Easy Cloner for my own use. I will try to eliminate

any bugs but can not promise to supply updates or much technical help.

If you have comments on Easy Cloner feel free to send them to

me and maybe you will see the effect of your suggestions in the next

version. My FAX number is (713) 792-4188. I also support a

shareware program called REFLIST that makes automatic reference lists

for scientific documents. If you want REFLIST send $30 in US currency

or US check to: E. Shillitoe, 5326 Dumfries Drive Houston TX 77096

USA.