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.