recombinant dna part h

... near the 5' end of the lacZ gene, the correct fusion (a tripartite gene) will have a Lac ÷ phenotype whereas the incorrect fusions will be Lac To confer the Lac ÷ phenotype, the DNA insert ... well as the pIFF8 vector itself have a Lac + phenotype). After cleavage with BssHII, the vector is treated with calf intestinal phosphatase (CIP) to remove the terminal 5'-phosphate...

Ngày tải lên: 11/04/2014, 10:26

... phosphate groups by incubation with nuclease-free alkaline phosphatase? 6,n~ The "¢ehicle DNA can then be ligated into a recombinant circle with the passenger DNA, or the passenger DNA ... short sequence, each repeat of which is probably less than 50 base pairs.92 Nevertheless, this DNA has been cloned with a high degree of sta- bility of the Xenopus DNA sequence, a9 Aft...

Ngày tải lên: 11/04/2014, 10:25

... of lambda DNA in a 15-hr reaction. 16 This phenomenon may be attributed to the viscosity produced by high concentrations of high molecular weight DNA (e.g., lambda DNA) , which can inhibit enzyme ... the products eventually are protein-free. It should be noted, however, that the presence of other nucleases will reduce the integrity of the product, whereas proteins tightly bound to th...

Ngày tải lên: 11/04/2014, 10:25

... DNA synthesis. 5 In contrast to the larger phage, the phage of this group usually contain circular DNA molecules. 6 The study of the DNA of these phage particles and the subsequent physical ... properties that aid their use as recombinant DNA vehi- cles: they do not lyse their host cells and they allow the packaging of larger than unit-length viral DNA. TM The two major compon...

Ngày tải lên: 11/04/2014, 10:26

... synthesis are supplied by the host. The DNA synthesis initiated by the action of the gene II protein (glIp) leads to both the replication of the ds molecule and the production of the ssDNA that ... strand nick within the IG region of the double-stranded replicative form (RF) of the phage DNA molecule at which DNA synthesis is initiated. Phage replication consists of three phases:...

Ngày tải lên: 11/04/2014, 10:26

... the poly(A) ÷ RNA prepared by this method can be converted to cDNA. If the number is considerably smaller than this, the RNA should not be used. Comments. Fresh tissue or healthy cells should ... Strand The HindlII-cut, C-tailed cDNA : mRNA-vector is cyclized by DNA ligase and the oligo(dG)-tailed linker DNA that bridges the C-tail and the HindlII end of the hybrid-vector. The RNA...

Ngày tải lên: 11/04/2014, 10:26

... least 5 U of methylase per microgram of DNA. Incubation is performed for 2 hr, and then the buffer is changed and the procedure repeated for 2 hr (or overnight if the methylase is highly purified). ... clones analyzed) and in high yield. Both analysis by SphI and BanI digestion led to two fragments each (the third BanI/HgiCI site is methylated in Escherichia coli and thus is not cl...

Ngày tải lên: 11/04/2014, 10:26

... edge of the microbead/TE droplet with the edge of a small piece of absorbent paper towel cut to the width of the tooth. Hold the towel against the edge of the tooth until all of the TE has been ... Emeryville, CA) and then expell them back to the pellet. They should pipette smoothly and have a consistency similar to that of a heavy oil. If they do not, they either (1) have clumps of micro...

Ngày tải lên: 11/04/2014, 10:26

... Run the bromphenol blue about 2 cm into the gel before examining the fluorescence. A successful reaction should have two bands, the ssDNA migrating slightly ahead of the dsDNA. 6. If ssDNA ... Institutes of Health, Bethesda, Maryland 20892 GEORGE M. CHURCH (14), Department of Genetics, Howard Hughes Medical Insti- tute, Harvard Medical School, Boston, Massachusetts 02115 JOHN A. CID...

Ngày tải lên: 11/04/2014, 10:27

... the DNA, e.g. with restriction endonucleases • Tie DNA molecules from different sources together with DNA ligase Restriction endonucleases generate ends that facilitate mixing and matching GAATTC CTTAAG GAATTC CTTAAG G CTTAA AATTC ... together Alternate method to join DNA: homopolymer tails Alternate method to join DNA: linkers Introduction of recombinant DNA into...

Ngày tải lên: 13/03/2014, 18:39