{from Thomas Seebacher  un691ts@genius.embnet.dkfz-heidelberg.de}
{Estimates the molecular weight of DNA restriction fragments or proteins}
{!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!   IMPORTANT    !!!!!!!!!!!!!!!!!!!!!!!!!!!!}
{********     Assumes the gel to run from top to bottom            *********}
{*********only the y-coordinates were used for the calculation*******}

{Count the number of bands in the reference lane you want to include as MW reference}
{Calibrate the gel first by entering the number of MW reference bands you want to include in your MW calibration curve}
{The macro gets the Y-coordinate for each band by mouse click and you must enter for each band their known molecular weight}

{the measurement counter was set to the position after the selected reference bands}


var
left,top,width,height,lower,upper,nm,n,m,x,y,z,MW,Pic1,Pic2,Pic3,i,j:integer;
nval, MWeight:string;


Procedure click;
begin;
SetCursor('cross');
repeat until button;
end;

Procedure error1;
begin;
putMessage('Two different  reference bands have the same y coordinate');
exit;
end;

Procedure Fwert;
begin;
n:=rMean[1];
if y<=rY[1] then
	begin
   {Out of range of  lower reference band};
if ((rY[2]-rY[1])=0) then error1;
   	MW:=rUser2[1]-(rUser2[2]-rUser2[1])*(rY[1]-y)/(rY[2]-rY[1]);
   MW:=exp(MW);
end;
if y>=rY[n] then
begin
   {Out of range of upper reference band};
if ((rY[n]-rY[n-1])=0) then error1;
   	MW:=rUser2[n]+(rUser2[n]-rUser2[n-1])*(y-rY[n])/(rY[n]-rY[n-1]);
   MW:=exp(MW);
end;
if (rY[1]< y) AND (y < rY[n]) then
begin
			i:=0;
  {selects the nearest reference bands};
			repeat;
					i:=i+1;
			until  rY[i]>y;
if ((rY[i]-rY[i-1])=0) then error1;
				MW:=rUser2[i]-(rUser2[i]-rUser2[i-1])*(rY[i]-y)/(rY[i]-rY[i-1]);
				MW:=exp(MW);
	end;
end;

Procedure Interpolate;
begin;
SetOptions( 'X-Y Center,User1');
{ShowMessage('Click mouse button to stop','\','\Y=  ',y:6:0,'\MW= ',MW:6:0);}
repeat;
   SetCursor('cross');
  GetMouse(x,y);
  Fwert;
  ShowMessage('Click mouse button to stop','\','\X= ',x:6:0,'\Y=  ',y:6:0,'\MW= ',MW:6:0);
  wait(0.3);
until button;
end;

Macro 'MW calibrate the gel with standards';
begin;
rMean[2]:=0;
ResetCounter;
SetCounter(1);
nval:=GetString('Number of reference bands=');
SelectWindow(WindowTitle);
n:=StringToNum(nval);
ShowMessage('n values=',n);
if n<2 then
begin
   PutMessage('You must enter at least 2 reference bands');
   exit;
end;
For m:=1 to n do
begin
     SetCounter(m);
     ShowMessage('Click on reference band ',m);
      click;
     GetMouse(x,y);
     rX[m]:=x;
{stores the y-coordinate of the reference band in rY array}
     rY[m]:=y;
     MWeight:=GetString('Molecular weight  = ');
     MW:=StringToNum(MWeight);
     rUser1[m]:=MW;
     rLength[m]:=MW;
{stores the log of the reference bands MW in the User2 array}
     rUser2[m]:=ln(MW);
    SelectWindow(WindowTitle);
    rMean[1]:=n;
end;
rMean[2]:=1;
SetCursor('arrow');
ShowMessage('Finished with reference band selection');
end;

Macro 'Continuous measurement  loop';
begin;
if (rMean[2]=0) then
begin;
  PutMessage('Enter  MW standards for your  gel');
   exit;
end;
SetCursor('cross');
repeat;
     Interpolate;
until button;
end;

Macro 'Measure the MW of selected bands';
begin;
if ( rMean[2]=0) then
begin;
  PutMessage('Enter  MW standards for your  gel');
   exit;
end;
n:=rMean[1];
nm:=n;
repeat;
     ShowMessage('Click on band of interest');
     SetCursor('cross');
     if (nm<>n) then ShowMessage('Press option key','\and click mouse button to stop','\X= ',x:6:0,'\Y=  ',y:6:0,'\MW= ',MW:6:0,'\Band#= ',rCount-n);
     Wait(1);
     click;
    GetMouse(x,y);
    nm:=nm+1;
    Fwert;
   SetCounter(nm);
    rUser1[nm]:=MW;
   rX[nm]:=x;
  	rY[nm]:=y;
  UpdateResults;
until KeyDown('option');
SetCounter(nm-1);
SetUser1Label('MW');
SetOptions( 'X-Y Center,User1');
ShowResults;
end;


end.