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

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.

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


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:

(Date:11/25/2015)... ... 25, 2015 , ... Jessica Richman and Zachary Apte, founders ... initial angel funding process. Now, they are paying it forward to other microbiome ... investments in the microbiome space. In this, they join other successful entrepreneurs-turned-angels ...
(Date:11/24/2015)... 2015 Halozyme Therapeutics, Inc. (NASDAQ: HALO ) will ... New York on Wednesday, December 2 at 9:30 a.m. ... and CEO, will provide a corporate overview. th ... at 1:00 p.m. ET/10:00 a.m. PT . Jim Mazzola ... a corporate overview. --> th Annual Oppenheimer Healthcare ...
(Date:11/24/2015)... CITY , Nov. 24, 2015 /PRNewswire/ - Aeterna ... request of IIROC on behalf of the Toronto Stock ... news release there are no corporate developments that would ... --> --> About ... . --> Aeterna Zentaris is ...
(Date:11/24/2015)... ... ... The Academy of Model Aeronautics (AMA), led by its Executive Council, has officially ... to represent the First–Person View (FPV) racing community. , FPV racing has exploded in ... racing and several new model aviation pilots have joined the community because of their ...
Breaking Biology Technology:
(Date:11/9/2015)... JOSE, Calif. , Nov. 9, 2015  Synaptics ... human interface solutions, today announced broader entry into the ... vehicle-specific solutions that match the pace of consumer electronics ... and biometric sensors are ideal for the automotive industry ... vehicle. Europe , ...
(Date:10/29/2015)... RESTON, Va. , Oct. 29, 2015 ... announced today that it has released a new version ... Daon customers in North America ... gains. IdentityX v4.0 also includes a FIDO UAF ... customers are already preparing to activate FIDO features. These ...
(Date:10/27/2015)... , Oct. 27, 2015 In the present ... of concern for various industry verticals such as banking, ... to the growing demand for secure & simplified access ... ,sectors, such as hacking of bank accounts, misuse of ... equipment such as PC,s, laptops, and smartphones are expected ...
Breaking Biology News(10 mins):