Navigation Links
UV Nicking of DNA in Agarose Gels for Enhanced Transfer and Detection of Megabase Size DNAs


Introduction
The transfer efficiency of large (>100 kb) DNAs from agarose gels to membrane is poor unless the DNAs are nicked prior to transfer. Although this has traditionally been accomplished with HCl depurination, UV irradiation (254 nm) provides a faster and more consistent alternative to HCl nicking. The GS Gene Linker UV chamber contains preprogrammed routines for many UV crosslinking and other controlled dosage applications, including UV nicking of pulsed field gels prior to transfer to membrane. The chambers unique sensor measures only the effective wavelengths of UV light, insuring reproducible results every time.


Results
High molecular weight DNA from a human HeLa cell line was cleaved with various restriction enzymes, resolved on the CHEF Mapper XA pulsed field electrophoresis system using the Auto Algorithm mode (50-750 kb), and irradiated with 60 mJoules of energy in the GS Gene Linker UV chamber after staining with ethidium bromide. The DNA was alkaline transferred to Zeta-Probe GT membrane, and probed with a human T cell receptor variable region (single copy gene) probe. As illustrated in Figure 1, bands ranging in size from 100 kb to greater than 700 kb were detected by the probe in the DNA lanes.


UV Nicking Protocol
Note: This protocol must be followed rigorously to obtain optimal results.
1. Following electrophoresis, stain the gel with 1.0 g/ml ethidium bromide (EtBr) for exactly 30 minutes with constant agitation. Use a fresh dilution of EtBr for each gel. Do not destain the gel.

2. Place the gel on a glass tray for tran sportation to the GS Gene Linker chamber, place the tray inside the GS Gene Linker chamber, and close the door.

3. Irradiate the gel with 60 mJoules of energy (nic program). The gel can now be photographed, but further exposure to UV light must be minimized (<10 seconds). The gel can be destained prior to photography if desired.

4. Soak the gel in 0.4 N NaOH, 1.5 M NaCl for 15 minutes. Transfer the DNA for at least 16 hours to Zeta- Probe GT membrane according to standard procedures, using 0.4 N NaOH, 1.5 M NaCl as the transfer solvent.

This protocol is for use with the GS Gene Linker UV chamber, with gels approximately 6 mm thick. If thicker gels are used, the staining period should be prolonged. DNA that is not stained with EtBr will not be nicked by UV irradiation, and will not transfer efficiently.


back to top
'"/>

Source:


Page: All 1 2

Related biology technology :

1. Simple Purification of DNA from Plasmid Minipreps, PCR Amplifications and Agarose Gels
2. Recovery of DNA from LMP Agarose1
3. Advantages of ReadyAgarose Precast Gels Over 96-Well Handcast Gels for High-Throughput Analysis, Rev A
4. Enhanced PCR Cloning System
5. Enhanced Amplification of Long Targets with PfuTurbo DNA Polymerase
6. Unique Enhanced DNA Polymerase Delivers High Fidelity and Great PCR Performance
7. Enhanced siRNA Delivery and Long-term Gene Silencing
8. Measuring Intracellular Enhanced Green Fluorescent Protein With the VersaFluor Fluorometer
9. Laser Capture Microdissection of Cells Labeled with Enhanced Green Fluorescent Protein
10. Increased Analyte Sensitivity through the Utility of Enhanced Mass-Resolution on the FinniganTSQ Quantum Discovery
11. Novel Bonding Chemistry Imparts Enhanced Polar Selectivity to TSK-GEL ODS-100V Reversed Phase Columns
Post Your Comments:
*Name:
*Comment:
*Email:


(Date:10/12/2017)... ... October 12, 2017 , ... ... analysis platform specifically designed for life science researchers to analyze and interpret ... Rosalind Franklin, who made a major contribution to the discovery of the ...
(Date:10/11/2017)... ... October 11, 2017 , ... The CRISPR-Cas9 ... enabling overexpression experiments and avoiding the use of exogenous expression plasmids. The simplicity ... for performing systematic gain-of-function studies. , This complement to loss-of-function studies, such ...
(Date:10/11/2017)... Alto, CA, USA (PRWEB) , ... October 11, 2017 , ... ... set to take place on 7th and 8th June 2018 in San Francisco, CA. ... policy influencers as well as several distinguished CEOs, board directors and government officials from ...
(Date:10/11/2017)... , Oct. 11, 2017  VMS BioMarketing, a leading provider ... nationwide oncology Clinical Nurse Educator (CNE) network, which will launch ... for communication among health care professionals to enhance the patient ... office staff, and other health care professionals to help women ... cancer. ...
Breaking Biology Technology:
(Date:4/18/2017)... , April 18, 2017  Socionext Inc., a global expert ... a media edge server, the M820, which features the company,s hybrid ... software provided by Tera Probe, Inc., will be showcased during the ... the NAB show at the Las Vegas ... ...
(Date:4/11/2017)... BEACH GARDENS, Fla. , April 11, 2017 ... identity management and secure authentication solutions, today announced ... contract by Intelligence Advanced Research Projects Activity (IARPA) ... for IARPA,s Thor program. "Innovation has ... onset and IARPA,s Thor program will allow us ...
(Date:4/6/2017)... Forecasts by Product Type (EAC), ... End-Use (Transportation & Logistics, Government & Public Sector, Utilities ... Generation Facility, Nuclear Power), Industrial, Retail, Business Organisation (BFSI), ... you looking for a definitive report on the $27.9bn ... ...
Breaking Biology News(10 mins):