Restriction enzyme digestion

Most often proteins are the bacterial biopolymers studied using MALDI MS either from fractions or whole cells. They are not the only isolated cellular biopolymers studied by MALDI, nor the first. Very soon after the introduction of MALDI there were a few reports of the analysis of bacterial RNA or DNA from bacterial fractions. One of the first applications of MALDI to bacteria fractions involved analysis of RNA isolated from E. coli,4 Other studies included analysis of PCR-amplified DNA,5 6 DNA related to repair mechanisms7 and posttranscriptional modification of bacterial RNA.8 While most MALDI studies involve the use of UV lasers, IR MALDI has been reported for the analysis of double stranded DNA from restriction enzyme digested DNA plasmids, also isolated from E. coli.9... 

Experiment 66 Separation of Restriction Enzyme Digestion Fragments via Horizontal Agarose Gel Electrophoresis... 

This experiment utilizes the restriction enzyme digests from Experiment 65. 

Using a micropipettor, add 40 jJL of each restriction enzyme digest from Experiment 65 to separate wells. 

T2. Todd, D. M., and Buccini, F. J., Apparent heparin interference with restriction enzyme digestion of genomic DNA. Clin. Chem. (Winston-Salem, N.C.) 39, 362-363 (1993). 

Eliminate Overlap by restriction enzyme digestion or by end-to-end PCR using 3 and 5 spedfic primers... 

Check of Plasmid DMAs by Restriction Enzyme Digestion in a 96-Well Plate... 

Flexible plate, 96 well, U-bottom without lid (BECTON DICKINSON, cat. No. 353911). There are several 96-well plates, but this product is easily handled especially for restriction enzyme digestion of a large number of DNA samples. 

Check successfiil cloning by restriction enzyme digestion and sequencing of the plasmid DNA see Note 7). 

Pick up a colony see Note 12) and check the plasmid by restriction enzyme digestion followed by agarose gel electrophoresis. 

Prepared plasmids are examined by a restriction enzyme digestion and an agarose gel electrophoresis. Insert sizes are examined and plasmids with an insert of an appropriate size are used for the next experiment. 

Restriction enzyme digestions should be performed by using a IO-20-fold dilution of the DNA. 

Mix 5 -12 pi PCR product or restriction enzyme digest reaction mixture with... 

It is recommended to include multiple positive controls (e.g. genomic DNA with heterozygous and homozygous mutations) and a negative control (water instead of template DNA) in each series. A validation of the procedure can be performed by sequencing some of the wild-type and mutant PCR products, as described in 8.2.3.5 Mutation Screening Restriction Enzyme Digest. 

Electrophoretic analyses of restriction enzyme digests and construction of a restriction enzyme map (see Experiment 15)... 

Reaction mixtures from restriction enzyme digestion may be analyzed directly by agarose gel electrophoresis. This technique combines high resolving power and sensitive detection to allow the analysis of minute amounts of DNA fragments. 

Standard restriction enzyme digest, X. phage DNA digested by roRI Constant-temperature water bath at 37°C... 

The HC and LC PCR products are pooled separately, phenol/chloroform-extracted, and ethanol-precipitated. Following resuspension and, where appropriate, gel purification of PCR products, the DNA is quantitated, before restriction enzyme digestion and cloning into thepComb3 vector. Gel purification (see below for methods) may be necessary when there are a high number of background bands in the PCR reaction. This tends to be more of a problem... 

Much like chromosomes, DNA fragments generated by restriction enzyme digestion of native DNA, when stained with DNA-specific... 

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