scholarly journals High-Efficiency T-Vector Cloning of PCR Products by Forced A Tagging and Post-Ligation Restriction Enzyme Digestion

BioTechniques ◽  
1997 ◽  
Vol 23 (5) ◽  
pp. 822-826 ◽  
Author(s):  
Helle Bielefeldt-Ohmann ◽  
David R. Fitzpatrick
Keyword(s):  
Restriction Enzyme ◽  
High Efficiency ◽  
Enzyme Digestion ◽  
Restriction Enzyme Digestion ◽  
Pcr Products

Isolation of large terminal sequences of BAC inserts based on double-restriction-enzyme digestion followed by anchored PCR

Genome ◽  
2000 ◽  
Vol 43 (2) ◽  
pp. 412-415 ◽  
Author(s):  
Zhong-Nan Yang ◽  
T Erik Mirkov
Keyword(s):  
Restriction Enzyme ◽  
Genomic Dna ◽  
Enzyme Digestion ◽  
Restriction Enzyme Digestion ◽  
Artificial Chromosomes ◽  
Bac Clones ◽  
Anchored Pcr ◽  
Pcr Products ◽  
Left And Right ◽  
The Right

Isolation of the terminal portions of genomic DNA cloned in bacterial artificial chromosomes (BACs) is an important step in map-based cloning, and several methods have been developed. Here, we present a new method based on double-restriction-enzyme digestion followed by anchored PCR. BAC DNA was digested with two enzymes: NotI and one of four enzymes (EcoRV, HpaI, StuI, or XmnI) that produce blunt termini. After dephosphorylation, these digestions were ligated to NotI- and EcoRV-digested pMSK, a new cloning vector developed in this work that is derived from pBluescript SK(+). PCR products representing the left- and right-terminal sequences of BAC inserts were obtained using a primer complementary to pMSK and a primer complementary to sequences in either the left arm or the right arm of the BAC vector pBeloBAC11. We have tested this method with 15 different BAC clones, and PCR products representing both the left- and right-terminal sequences have been obtained from all 15 BAC clones. This method is simple, fast, reproducible, and uses the same set of primers for any restriction enzyme used. With some modifications, it can also be used for isolating the terminal portions of genomic DNA cloned in yeast artificial chromosomes and P1-derived artificial chromosomes. Key words: BAC, anchored PCR, terminal sequence isolation, chromosome walk.

The stability of maize nuclei to restriction enzyme digestion differs with transcriptional activity

Caryologia ◽  
1993 ◽  
Vol 46 (1) ◽  
pp. 63-69 ◽  
Author(s):  
Valeria Mirkova ◽  
Maria Ivanchenko ◽  
Lubomir Stoilov ◽  
Jordanka Zlatanova
Keyword(s):  
Restriction Enzyme ◽  
Transcriptional Activity ◽  
Enzyme Digestion ◽  
Restriction Enzyme Digestion ◽  
The Stability

Identification of the CD32/FcγRIIc-Q13/STP13 polymorphism using an allele-specific restriction enzyme digestion assay

2001 ◽  
Vol 258 (1-2) ◽  
pp. 85-95 ◽  
Author(s):  
D Metes ◽  
A.A Gambotto ◽  
J Nellis ◽  
A Ruscin ◽  
A.M Stewart-Akers ◽  
...  
Keyword(s):  
Restriction Enzyme ◽  
Enzyme Digestion ◽  
Restriction Enzyme Digestion ◽  
Allele Specific ◽  
Specific Restriction

Resistance of Tumor-Derived DNA to Restriction Enzyme Digestion

1990 ◽  
Vol 8 (2) ◽  
pp. 169-172
Author(s):  
Joan Lee Parkes ◽  
Frank C. Hubbard ◽  
Arthur Penn
Keyword(s):  
Restriction Enzyme ◽  
Enzyme Digestion ◽  
Restriction Enzyme Digestion

Glass-Polydimethysiloxane Hybrid Microthermostat for Restriction Enzyme Digestion

2007 ◽  
Vol 544-545 ◽  
pp. 335-338
Author(s):  
Yong Jun Ko ◽  
Dae Jin Kim ◽  
Woong Cho ◽  
Yoo Min Ahn ◽  
Seung Yong Hwang
Keyword(s):  
Conventional Method ◽  
Restriction Enzyme ◽  
Gel Electrophoresis ◽  
Low Cost ◽  
Reaction Chamber ◽  
Enzyme Digestion ◽  
Pyrex Glass ◽  
Restriction Enzyme Digestion ◽  
Good Biocompatibility ◽  
Pdms Replica

This paper reports a low-cost microthermostat that is able to maintain a constant temperature necessary for restriction enzyme digestion. Polydimethylsiloxane (PDMS) and Pyrex glass were used to make the microthermostat, because PDMS is a cheap and mass-producible material and both PDMS and glass have very good biocompatibility compared to the more commonly used silicon. A heater made of Au wiring patterned on Pyrex glass was used to control the temperature. A PDMS replica molding technique was used to fabricate a reaction chamber with 3.6 μl capacity. Restriction enzyme digestion was performed by using the fabricated microthermostat and by a conventional method. Then, using gel electrophoresis, we compared results between the microthermostat and conventional methods. It was found that restriction enzyme digestion using the microthermostat required 5 min of heating.

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