gel.doc update 10/29/90 GEL I. Function: Given the lengths and mobilities of a set of molecular weight standards on a gel, this program will estimate the sizes of unknown fragments whose mobilities are known, using the least squares approach found in: Schaffer & Sederoff, ANALYTICAL.BIOCHEM. 115,p113-122 (1981) II. Menus GEL begins by asking for a filename for output, and then the user is placed in the main menu. It is best to conceptualize of the main menu as performing those tasks in which GEL communicates with the operating system, such as opening and closing files, or writing output. The status line tells the current output filename (the printer in the example below). _____________________________________________________________________ GEL MAIN MENU _____________________________________________________________________ Output file: prn _____________________________________________________________________ 1) Type in a set of standard fragments 2) Edit values of standard fragments 3) Add fragments 4) Delete fragments 5) Calculate sizes of unknowns (output to screen) 6) Calculate sizes of unknowns (output to file) 7) Open a new output file _____________________________________________________________________ Type the number of your choice (0 to quit program) 1 Type in the size of each fragment and distances migrated, one fragment at a time. Press after each fragment. Type 0 0 when finished Length Mobility --------+------------ (* User types in standard frags. *) 23.7 0.4 9.46 0.7 6.75 0.85 4.26 1.15 2.26 1.6 1.98 1.75 0.58 2.7 0 0 After the fragments have been typed in, you may choose option 2 in the main menu to edit fragments. The menu is shown below: _______________________________________________________________________ Standard fragments: Length Mobility (* The current fragment *) 1) 23.700 0.400 (* list is displayed when *) 2) 9.460 0.700 (* editing, adding or *) 3) 6.750 0.850 (* deleting fragments. *) 4) 4.260 1.150 5) 2.260 1.600 6) 1.980 1.750 7) 0.580 2.700 _______________________________________________________________________ Type number of a fragment you wish to change, or 0 if all are correct. 0 When you are satisfied with the markers typed in, choose option 5 do determine the sizes of the unknowns. Type title to appear on output ( for blank): pI inserts, using Lambda Hind3 standards Type an identifier (<=10 letters) for an unknown fragment ( to quit) pI230 Distance migrated? 2.7 Type an identifier (<=10 letters) for an unknown fragment ( to quit) pI259 Distance migrated? 2.95 Type an identifier (<=10 letters) for an unknown fragment ( to quit) pI286 Distance migrated? 2.35 Type an identifier (<=10 letters) for an unknown fragment ( to quit) (* The output appears on the screen as shown below: *) pI inserts, using Lambda Hind3 standards STD LEN DIST PRED LEN DEVIATION %DEV C[I] 23.70 0.400 23.79 -0.086 -0.364 5.791 9.46 0.700 9.28 0.178 1.876 5.905 6.75 0.850 6.85 -0.095 -1.416 5.746 4.26 1.150 4.21 0.045 1.058 5.855 2.26 1.600 2.34 -0.081 -3.618 5.694 1.98 1.750 1.95 0.025 1.272 5.851 0.58 2.700 0.57 0.009 1.615 5.835 M0= 1.7225060E-01 L0= -1.7282030E+00 CCAP= 5.8108870E+00 SC= 7.1956960E-02 SD= 1.1990510E-01 UNKNOWN FRAGMENTS: FRAGMENT DISTANCE PREDICTED LENGTH pI230 2.70 0.571 pI259 2.95 0.364 pI286 2.35 0.940 The you may edit, add, or delete fragments and re-calculate the standard curve function. For example, to add a fragment, choose option 3. You will be prompted for a position in the list, before which you wish the new standard to be added. To add a fragment to the end of a seven-fragment list, type 8 as a response. _______________________________________________________________________ Standard fragments: Length Mobility 1) 23.700 0.400 2) 9.460 0.700 3) 6.750 0.850 4) 4.260 1.150 5) 2.260 1.600 6) 1.980 1.750 7) 0.580 2.700 _______________________________________________________________________ Add a fragment before which number? (0 to quit) 8 Type new fragment length and mobility 0.125 3.5 _______________________________________________________________________ Standard fragments: Length Mobility 1) 23.700 0.400 2) 9.460 0.700 3) 6.750 0.850 4) 4.260 1.150 5) 2.260 1.600 6) 1.980 1.750 7) 0.580 2.700 8) 0.125 3.500 _______________________________________________________________________ Add a fragment before which number? (0 to quit) 0 To delete a fragment, choose option 4. You will be prompted for the number of a fragment to delete. III. Constants MAXFRAGS and MAXLINE are defined in the constant definition part of the main procedure of GEL. To change them, it is necessary to change their values in the Pascal text and re-compile. MAXFRAGS The maximum number of standard fragments. Set by default to 50. MAXLINE The maximum length of a variable of the type LINE, used here only for the title to be printed on output. Set by default to 80. IV. What the output means Although the output is mostly self-explanatory, several features will be described briefly. The columns STD LEN and DIST refer to the actual lengths and migration distances typed in by the user. PRED LEN is the predicted length that a fragment should have, based on the actual distance migrated, as determined by linear regression, using all the standards. DEVIATION is the difference between the predicted length and the actual length, and %DEV is the same figure, expressed as a percentage of the actual length. C[I] is explained in the reference, giving an indication of how closely each data point fits to the best straight line. C[I] values may vary widely. V. Usage Notes 1. Fragment sizes may be typed in as either length (kb or bp) or molecular weight. Distances migrated on the gel may be entered using any units. The only limitation is that, for any given set of data points, usage must be consistant. 2. This program requires at least 4 standard fragments. 3. GEL provides a number of useful statistical guides for checking the data. For any given standard, expect a low value for %DEV (<=5). This tells you the confidence interval in a given area of the gel. The best indicator of the data as a whole is SD, the standard deviation of fragment lengths. This statistic is affected by both the range of standard fragments, and the goodness of fit. 4. There are sometimes cases in which you may have more bands than expected in your standard lane. These extra bands may be partial digest bands, supercoiled or relaxed circular DNA bands, or, on denaturing gels, in which DNA fragments are used as size standards for RNA size determinations, double-stranded bands. GEL is the ideal tool for sorting out which fragments are which. To solve this problem, make a best guess as to which fragments are which, and type them in. If SD is low, and all of the standards have a low %DEV, you have probably made the right guess. If not, the incorrect fragment will have a high %DEV. Experiment with one band at a time, keeping all the other standards constant, until you have found a band whose distance migrated best fits the rest of the data. 5. Obviously, the accuracy of your measurements depends on the accuracy of the correct sizes of your standards. Try to always use standards for which the complete DNA sequence is known, such as Lambda phage or pBR322. 6. In many cases it is more convenient to begin the program with output going to the screen, rather than to the printer or a file. This enables you to quickly try different combinations of bands to clear up ambiguities, as discussed in note 4, and then send output to the printer for a final printout.