Automation of 96 Gel Column Array for SizeSize-Exclusion & Desalting of
Chlorophyll on VERSA 10 Workstation
Sikander Gill1 PhD, Rajwant Gill1 PhD, Barry Yelenik2 and Dong Liang1 PhD
Aurora Biomed Inc., Vancouver, BC, Canada1, EMP Biotech, Howell, NJ, USA2
Contact: Barry Yelenik e-mail: [email protected] or Sikander Gill e-mail: [email protected]
I. Summary
Gel filtration is widely used in diverse downstream bioprocesses
including DNA size separations, removal of dyes, salts and other low
molecular mass reagents, purification of >10 bp oligonucleotides, and
buffer exchange in downstream bioprocesses. CentriPure N96, a gel
array was automated on a very compact VERSA 10 Workstation. The
automation allows desalting of 96 samples in 4 minutes including
sample addition, pre-run and elution. Subsequent washing and
equilibration of the columns automatically continues for the userdefined volume or duration. The eluted samples are auto-incubated at
the user-defined temperature in the range of 2-90°C. A total of 104
tips (1000 or 200µl) are used.
The results show that the automated operation performed with
consistency, high throughput, and accuracy did not suffer from any
detectable cross-contamination indicating no dripping or hanging
drops from the columns into eluted samples. The resulted 94.4%
recovery was found to be equivalent to that of manual process
(94.35%).
II. Introduction
Gel filtration, a non-adsorptive chromatography technique separates
molecules on the basis of molecular size exclusion. It is widely used
for DNA size separations, purification of sensitive biomolecules,
removal of low molecular mass reagents, adjusting of ionic strength of
a protein or DNA solution, removal of dyes, purification of >10 bp
oligonucleotides, desalting, and buffer exchange in diverse
downstream bioprocesses1-3. However, manual operation of this
process faces many challenges as outlined in table 1 and thus
requires automation.
Table 1
IV. Materials & Methods
V. Results & Discussion
Automation of desalting of chlorophyll from plant extract has been
presented below:
The validation of the CentiPure N96 Gel Filtration (www.empbiotech.com) was
conducted on VERSA 10 Workstation (www.Aurorabiomed.com) with 8-Channel
liquid handling, and 6 deck position configuration as follows:
1.Samples: 96 samples of the followings were purified:
1. a. Hyladder DNA, and b. Plant extract (DNA, Protein, and chlorophyll)
2. Deck layout: The deck was configured as shown in Figure 1.
3. Protocol: Automated steps were performed as follows (Figure 2):
a. Equilibration of gel columns with distilled water:
1. Addition of 5 ml by repeating cycles of 300µL.
2. The flow out waste liquid was collected in the underneath collection module
which was connected with a tubing to a waste bottle kept under the table.
b. Sample addition: Sample of plant extract (25 or 50 or 100 µl) were added
to the columns with a user-defined controlled low dispense speed to minimize
disturbance to the sample distribution in the gel
c. Pre-run liquid addition: A calculated volume of distilled water was followed the
sample addition so that sample + Pre-run volume = 350 µl.
d. Moving of the CentriPure N96 Gel Array: The Gel array was automatically
moved between wash module and elution module ensuring the followings:
a. No dripping
b. No hanging of drops
e. Elution: 300 µl elution reagent (distilled water) was added to each
columns.
4. Cross-contamination This test was designed as a checker-board with alternating
plant extract samples and distilled water.
5. Manual process: The manual process was carried after the automation for
comparative evaluation.
6. Analysis: The Hyladder DNA, DNA, protein and chlorophyll in plant extract was
scanned (240-900nm) and OD260, OD280, and OD680, respectively was analyzed on
Epoch Microplate Spectrophotomete (www.biotek.com)
Figure 1: a. VERSA 10 Workstation b. Deck Layout
1.
Recovery of chlorophyll from plant extract: The objective of this experiment was to examine whether the
workstation can achieve recovery of eluted molecules comparative to that of manual process. The average
recovery of both the manual and automated process was comparable (manual process: 94.35% vs automated
94.4%. A CV% of the inter-channel (1.3-6.4%, n=12) and intra-channel (2.1-6.2%, n=12) recovery was also
compared for the automated process (Table 2).
Table 2
Table 3
2. Cross-contamination test: This experiment was designed to test any cross contamination during the automated
process due to cross contaminating liquid handling due to wrong X-Y precision movement of the arm, splashing
or over flowing of columns or eluted sample plate, or due to dripping or hanging drops from the bottom ends of
columns. The OD680 data from the eluted samples in checker-board format indicated no cross contamination
during the automated process (Table 3).
Table 4
Table 5
3. Throughput: The flow down of the samples and reagents through the columns was carried by gravitation flow
while the system offers options from mild to strong vacuum (negative pressure) or positive pressure. The
gravitation flow was recorded as 4.46 min/ml. The total duration of steps of the process is presented in Table 4.
VI. Why VERSA 10 Workstation?
In addition to the efficient automation of gel filtration, VERSA 10 Workstation is a very compact (L 57cm x W 45cm
x H 54) to fit on standard lab tables with economical spacing and offers various additional applications with or
without add-on modules (Table 5). The system offers a throughput of 96 samples within 21 minutes and a total of
104 tips (1000 or 200µl). The results show that the automated operation performing with consistency, high
throughput, and accuracy, handling of 96 well or 384 well formats can bring savings on time, energy and add
confidence in the accuracy of results. This system with high efficiency air filter hood equivalent to a small ultra
clean workbench, equipped with optional modules like shaker, cooling-heating incubator(s), and with smooth
liquid handling, effectively avoids cross contamination which is highly essential for diagnostic and other
applications.
Keeping this in view, a 96 gel column array, CentiPure N96 Gel
Filtration was automated on VERSA 10 Workstation.
III. Objectives
1.
2.
3.
4.
5.
To validate the separation of the following molecules
a.
DNA from blue dye
b.
Chlorophyll and salts from plant extract
c.
DNA from plant extract
d.
Protein from extract
Optimal size of sample
Optimal recovery
Any cross-contamination
Accurate reagent dispensing
VIII. References
Figure 2: Automated steps of the gel filtration process
1. Escriou etal., Triple helix formation on plasmid DNA determined by a size-exclusion chromatographic method.
Anal Biochem.1997; 248(1):102-110.
2. Almadaly etal., Desalted and lyophilized bovine seminal plasma delays induction of the acrosome reaction in
frozen-thawed bovine spermatozoa in response to calcium ionophore. Theriogenology. 2015; 83(2):175-185.
3. Xiong etal., Glucomannan microspheres for low-cost desalting of protein solution. Carbohydr Polym. 2014;
111:56-62
*Correspondence should be addressed to Sikander Gill e-mail: [email protected], Aurora Biomed Inc., 1001 E. Pender St., Vancouver, BC, Canada, V6A 1W2, Tel: 1-604-215-8700, Fax: 1-604-215-9700www.aurorabiomed.com