Waters Gen-Pak FAX Columns - Manual (Page 5)

[ Care and Use ManUal ]
Gen-Pak FAX Columns 5
V. eXAMPLe UsInG HAe III PLAsMID DIGesteD DnA
This section shows an example of how to develop a method using
Gen-Pak FAX columns. The example in this chapter can serve as a a
model for adapting this technique to the requirements of a particular
sample.
The example describes the evolution of the separation of a restriction
enzyme digest, the Hae III digest of ØX 174 RF DNA. This DNA sample
contains 11 fragments ranging in size from 72 to 1353 base pairs.
Table 1: Number of Base Pairs in Each Fragment
a. Initial Gradient
Chromatography is initally performed using a relatively steep
ionic strength gradient. As Figure 3 shows, these conditions do not
completely resolve the 234, 271, 281, and 310 (peaks 4-7) nor the
603, 872, 1078, and 1353 (peaks 8-11) base pair fragments.
Figure 3: Initial Separation uf 2 μg of Hae III Digest of ØX 174 RF
DNA at 30°C using a Steep Salt Gradient
b. Gradient Optimization
Resolution can often be improved by systematic adjustment of
gradIent conditions. In the current example, examination of the inital
chromatorgram indicates a more shallow gradient is required.
c. Calculating NaCl Concentration
It is possible to calculate the approximate NaCl concentration
required to elute the first (72bp) and last ( 1353 bp) restriction
fragments from the data obtained in the inital chromatography.
First, determine the system delay volume from the point of gradient
formation to the detector cell.
d. Calculating System Delay Volume
To calculate the system delay volume from point of gradient
formation to detector cell:
1. Remove the column from the system, insert a union, and set the
detector to 260 nm.
2. Flush the pump and solvent lines with eluent A ( Milli-Q water)
3. Start the flow of eluent B (0.01% acetone in Milli-Q water)
4. Monitor the detector output until it rises to the new, higher value
of about 0.032 absorbance units if a cm detector cell is used.
The system delay volume corresponds to the volume that has flowed
from the start of eluent B to the midpoint of the absorbance rise plus
the excluded column volume of 0.6 mL.
e. Using a More Shallow Gradient
The system delay volume for this instrument was 7.5 mL,
corresponding to 10 minutes at 0.75 mL/minute.
Under inital conditions, elution occurs as follows:
• The 72 base-pair fragment elutes at 22 minutes, reflecting
the NaCl concentration at 12 minutes in the gradient table of
0.56 M NaCl
• The 1353 base-pair fragment elutes at 0.65 M NaCl
Figure 4 shows the results of changing to a more shallow NaCl
gradient, from 0.54 to 0.67 NaCl, over the same 30 minute time
interval. As this figure shows, the change significantly imrproved the
resolution.
Fragment Base Pairs
1 72
2 118
3 194
4 234
5 271
6 281
7 310
8 603
9 872
10 1078
11 1353
Absorbance at 260 nm
Minutes
0
10 35
1 2
3
4
5-7
8-11 0.2
Column: Gen-Pak ô FAX (4.6 mm x 100 mm)
Buffer A: 25 mM Tris/Cl, 1mM EDTA, pH 8.0
Buffer B: 25 mM Tris/Cl, 1 mM EDTA, 1.0 M NaCl, pH 8.0
Gradient: 30 to 100% B in 30 min, linear
Flow: 0.75 mL/min
Temperature: 30˚C