Please read carefully INTRODUCTION before starting ElQuant. It is recommended to combine this program with Virtual electrophoresis program

1. ABOUT ELQUANT

DNA Size

ElQuant program accepts DNA fragment lengths described in the Table 1

Type of the gel:	The program works with the following size range (bp):	Working range (bp):
Spreadex EL 300	20 - 280	10 - 300
Spreadex EL 400	20 - 360	15 - 400
Spreadex EL 500	40 - 400	20 - 500
Spreadex EL 600	40 - 500	40 - 600
Spreadex EL 800	60 - 700	60 - 800
Spreadex EL 1200	80 - 1000	80 - 1000
Poly(NAT) 6%	80 - 1000	80 - 20 000
Poly(NAT) 9%	80 - 1000	40 - 2000
Poly(NAT) 12%	60 - 800	20 - 800
PCR CheckIT with EB	100-1000	80 - 5000
PCR CheckIT	100-1000	80 - 5000

Separation of any two fragments can be simulated in the size range indicated.
Long fragments, up to 20,000 bp, ca be separated on 6% Poly(NAT) gels, but the electric field strength should be 3V/cm.

Runing Time

The values calculated by ElQuant are valid only at the specified temperatures (20°C or 55°C). It is not possible to calculate exact running time without keeping the temperature constant during the entire run. Not recommended are the runs longer than 4 hours at 55°C (due to excessive water evaporation) and longer than overnight (18h) at 20°C. In general, one should select the shortest running time that provides adequate separation.

To compensate for the temperature fluctuations during electrophoresis with equipment of other manufacturers, it is prudent to select a running time that is 20% lower than the value calculated by ElQuant at 20°C, in order to prevent the bands from migrating out of the gel.

Electric Field Strength

The Running Time values calculated by ElQuant are valid only at the electric field strength of 10 V/cm. In submarine electrophoresis, the electric field strength equals the applied voltage divided by the distance between electrodes. The runs are performed at constant voltage. The amperage is always set to maximum and remains constant during the entire run under steady state conditions achievable in Elchrom SEA 2000 apparatus. Electric field strength lower than 10 V/cm may provide better separation of some fragments on Poly(NAT) gels.

2. HOW TO USE ELQUANT

Gel resolving power determines the distance between two separated bands. The higher the resolving power, the larger is the distance on a given gel length. Minimal separation distance required for clear differentiation of two bands is 1 mm when using photographic recording.

To determine the running time necessary to separate two fragments, enter the size of the shorter fragment in the lower, and the size of the longer fragment in the upper field. Choose the temperature and the type of the gel (check the recommended gel type in Section 3). Click Result: Indicated are: the running distance of the shorter fragment, distance between the two fragments, and the running time. Different options are offered. Check which option satisfies your need and choose the adequate size (format) of the gel. To make this choice easier, the maximum running distance for the shorter fragment indicates at the same time the maximum running distance (length) of different gel formats (check Section 3). To be sure that the shorter fragment will not run out of the gel, always shorten the running time chosen, for approx. 10 min.

Separation of More Than Two Fragments

If there are more than 2 fragments present in the sample, calculation is done in two steps.
First enter the sizes of the two fragments that are the closest in the size to see the running time necessary to resolve them. Note the running time value at which the distance between the two bands (shown in the middle column) is at least 1 mm. Secondly, enter the size of the shortest and the longest fragment. Click Result that will show maximum running distance for the shortest fragment and the corresponding running time. Check that the actual running time is the same or longer than the running time necessary for separating the two closest bands and at the same time not longer than the maximum running time for the shorter fragment. These two values will determine which gel format and the type of the gel will be optimal in resolution, running time and the throughput for your analysis.

3. DIFFERENT GEL TYPES AND GEL SIZES (FORMATS)

Gel Selection

Elchrom Scientific produces ready-to-use gels of three different (non-agarose non-acrylamide) compositions. Spreadex gels are used for the most demanding separations in narrow size ranges (1% difference in size can be separated). Each Spreadex gel is characterized by an Exclusion Limit (EL). DNA fragments of the size above the Exclusion Limit remain in the sample well. Poly(NAT) gels cover broad size range with high resolution of 2.5-3% difference in size. PCR CheckIT gels are recommended for fast runs and easy DNA recovery (5% of the size can be resolved). PCR CheckIT gels with and without EtBr are available. DNA fragments are retarded in gels with EtBr.

Precast GMA (Gene Mutation Analysis) gels for SSCP and heteroduplex analysis separate DNA fragments mostly on the basis of their sequence, and therefore it is not possible to provide exact data on the running time and separation.

Gel Length

The gel length available for separation on Elchrom Scientific ready-to-use gels varies from 8 to 87 mm. The length is related to the number of sample wells. Choosing the shortest gel length that provides adequate resolution, will increase the throughput and reduce the cost per sample.

The number of sample wells on Elchrom Scientific ready-to-use gels varies from 8 to 400. The Mini gels have 8 and 12 sample wells, the Wide Mini have 2x13, 4x13, 2x25, 4x25, 2x104, 2x200 sample wells (S). The S-2x104 and S-2x200 gels have eight rows of sample wells.

Format of the gel	Length in mm	Number of sample wells
Mini with 12 sample wells Mini with 8 sample wells	56 87	12 8
Wide Mini S-2x13	87	2x13
Wide Mini S-4x13	40	4x13
Wide Mini S-2x25	87	2x25
Wide Mini S-4x25	40	4x25
Wide Mini S-2x104L	16	2x104
Wide Mini S-2x104	8	2x104
Wide Mini S-2x200	8	2x200

DNA Concentration

Optimal DNA concentration depends on the detection method. For Ethidium Bromide staining, the DNA amount per band should be 2-20 ng, for SYBER Gold (or SYBER Green) staining 0.4-4 ng.

Overloading impairs resolution. The samples containing unknown amount of DNA should be loaded at two dilutions, one being 10-fold lower than the other.

Optimal loading volume is:	4-8 µl
Maximum loading volumes are:      Mini gel      Wide Mini S-2x13, S-4x13, S-2x104      Wide Mini S-2x25, S-4x25, S-2x104L, S-2x200	16 µl 25 µl 14 µl

4. DISCLAIMER

THE SOFTWARE IS PROVIDED "AS-IS" AND WITHOUT WARRANTY OF ANY KIND, EXPRESS, IMPLIED OR OTHERWISE, INCLUDING WITHOUT LIMITATION, ANY WARRANTY OF MERCHANABILITY OR FITNESS FOR A PARTICULAR PURPOSE.

IN NO EVENT SHALL ELCHROM BE LIABLE FOR ANY SPECIAL, INCIDENTIAL, INDIRECT OR CONSEQUENTIAL DAMAGES OF ANY KIND, OR ANY DAMAGES WHATSOEVER RESULTING FROM LOSS OF USE, DATA OR PROFITS, WHETHER OR NOT ADVISED OF THE POSSIBILITY OF DAMAGE, AND ON ANY THEORY OF LIABILITY, ARISING OUT OF OR IN CONNECION WITH THE USE OR PERFORMANCE OF THIS SOFTWARE.

ELCHROM RESERVES THE RIGHT TO MAKE MODIFICATIONS OF THIS SOFTWARE WITHOUT PRIOR NOTICE.