Agarose Gel Electrophoresis

Leonard Thou. Davis Ph.D. , ... James F. Battey M.D., Ph.D. , in Bones Methods in Molecular Biology, 1986

DESCRIPTION

Agarose gel electrophoresis is used to resolve DNA fragments on the ground of their molecular weight. Smaller fragments drift faster than larger ones; the distance migrated on the gel varies inversely with the logarithm of the molecular weight. The size of fragments tin therefore be determined by calibrating the gel, using known size standards, and comparing the distance the unknown fragment has migrated. This technique can be used to resolve complex DNAs (i.e., genomic Deoxyribonucleic acid) for Southern blot analysis or to resolve simpler digests of bacteriophage and plasmid clones for RE site mapping and blotting.

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Agarose gel electrophoresis

Khalid Z. Masoodi , ... Rovidha Saba Rasool , in Avant-garde Methods in Molecular Biological science and Biotechnology, 2021

Abstract

This chapter firstly gives a brief introduction to the method of electrophoresis. Information technology and so emphasizes the importance of agarose gel electrophoresis in terms of the separation and analysis of macromolecules like Dna, RNA, and protein on the basis of their molecular weights. The gel works the aforementioned way as the sieve. The molecules split up due to their characteristic accuse through the sieve. Thus, within the pool of molecules, size separation is achieved across the gel. In this manner, researchers can place the segments and tin compare the Deoxyribonucleic acid of unlike species. Furthermore, the chapter mentions the materials and types of equipment required to carry out agarose gel electrophoresis along with their importance. A footstep-by-step protocol volition help the students and researchers to follow the procedure efficiently and finer.

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Quantitation of Nucleic Acids

Frank H. Stephenson , in Calculations for Molecular Biology and Biotechnology, 2003

Estimating Deoxyribonucleic acid Concentration on an Ethidium Bromide-Stained Gel

Agarose gel electrophoresis is commonly used to separate Deoxyribonucleic acid fragments following restriction endonuclease digestion or PCR amplification. Fragments are detected by staining the gel with the intercalating dye, ethidium bromide, followed by visualization/photography nether ultraviolet low-cal. Ethidium bromide stains DNA in a concentration-dependent manner such that the more than DNA present in a ring on the gel, the more intensely it volition stain. This relationship makes information technology possible to estimate the quantity of Dna present in a band through comparing with some other band of known Deoxyribonucleic acid corporeality. If the intensities of two bands are similar, then they incorporate similar amounts of Dna. Ethidium bromide stains single-stranded DNA and RNA only very poorly. These forms of nucleic acrid will not give reliable quantitation by gel electrophoresis.

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Nucleic Acid

Buddhi Prakash Jain , ... Shweta Pandey , in Protocols in Biochemistry and Clinical Biochemistry, 2021

Rationale

Agarose gel electrophoresis is an easy and efficient method to separate, identify, and purify the Dna molecules. The location of Deoxyribonucleic acid can besides be adamant with this method by staining with fluorescent dyes, which can observe up to 20  pg of double-stranded DNA by examination of the gel under UV. Agarose gels have relatively lower resolution power than polyacrylamide gels merely a greater range of separation.   In this process, 50 bp to several megabases of DNA can be resolved in agarose gel (about suited for fifty–20,000 bp). Agarose is a linear polymer, it comprises alternate d- and l-galactose joined by α(one-3) and β(1-4) bonds with anhydro bridge between iii and 6 positions. It gelatinizes to class a three-dimensional mesh of channels of size ranging from 50 to ≥   200   nm. The rate of migration of the Dna sample depends on various factors as stated in the previous chapter. Ane of the factors is the size of the Deoxyribonucleic acid sample.

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Electrophoretic techniques

Apratim Maity , ... Chukwuebuka Egbuna , in Analytical Techniques in Biosciences, 2022

4.4.ii DNA electrophoresis

Agarose gel electrophoresis is widely used for separation of DNA and RNA samples in events like restriction fragment analysis, polymerase concatenation reaction production assay, checking the integrity of genomic Deoxyribonucleic acid, and purification of nucleic acids. DNA and RNA are negatively charged and during electrophoresis, the side of the gel having wells is placed near the cathode. The rate of motion of linear Dna is inversely proportional to the log x of its molecular weight. Tris-acetate-EDTA or tris-borate-EDTA (TBE) buffers are used for DNA/RNA electrophoresis. Bromophenol blueish or xylene cyanol are used equally loading dye and mixed with the nucleic acid sample and then that, the electrophoretic run tin be tracked till these dyes move near the other end.

The gels are visualized by exposing it to ultraviolet (UV) light after staining with ethidium bromide or SYBR green. The dyes are embedded in the gel by adding them to the gel before casting. Alternatively, the gel tin be stained after electrophoresis. The dyes are mutagenic and hence should exist handled with proper precaution. Intendance should besides be taken during visualization in UV transilluminator, so that the exposure of the person to these harmful rays tin can exist prevented. For documentation purpose, the photo of the gel tin be taken using gel documentation arrangement.

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RIFT VALLEY FEVER VIRUS INTRACELLULAR RNA: A FUNCTIONAL ANALYSIS

M.D. Parker , ... J.M. Dalrymple , in Segmented Negative Strand Viruses, 1984

RNA Part

Agarose gel electrophoresis of radiolabeled RNA extracted from infected cells revealed an RNA of approximately 300,000 daltons, in improver to the three RNAs which migrate to the positions of the genome segments L, M and S ( fig. 3).

Figure 3. Rift Valley fever virus specific intracellular RNA. Infected Vero cells were treated with 10 μg/ml Actinomycin D at 4.five hours postinfection and pulsed with 300 μCi/ml 32P-orthophosphate from hours v through 7. Cells were lysed in 80 mM KCl, xx mM tris-HCl, pH 7.four, five mM MgCltwo, i% NP-40. The cytoplasmic RNA was extracted with phenol: chloroform. Radioactive virus was prepared as described in the legend to Figure 1. Electrophoresis was as described (7). Virion RNA (Lane A); Intracellular RNA (Lane B).

In society to farther characterize these RNAs, lysates of infected cells were fractionated by CsCl centrifugation (8), yielding a pellet rich in ribosomal RNA and a peak of RNA at a density of 1.29, characteristic of virion ribonucleoproteins (RNP). Agarose gel electrophoresis of the RNA in the RNP fraction yielded merely genome sized RNAs (fig. four). The pellet also independent three virus-specific species of RNA. One migrated slightly ahead of the M segment found in the RNP, some other migrated precisely with the S segment seen in the RNP fraction and the third was the 300,000 dalton RNA. Because the pelleted material consisted largely of polysomal associated RNA (9), information technology was expected that the virus-specific RNA in the pellet would be of positive polarity and would therefore hybridize to virion RNA. Unlabeled, RVF virus-infected cells were fractionated on CsCl and both RNP and pelleted RNA fractions were analyzed by Northern blotting. Probe was prepared by labeling a partial RNAse T1 assimilate of virion RNA with polynucleotide kinase and 32P-ATP. Virion RNA probes hybridized to all 3 bands in the RNA extracted from intracellular ribonucleoproteins and to the three bands in the pelleted RNAs (fig. iv). An identical pattern of hybridization was obtained when RNA from the intracellular ribonucleoproteins was utilized every bit probe (data not shown). These results indicate that intracellular ribonucleoproteins contain RNA of both plus and minus polarity and that the CsCl gradient pellets contain plus stranded RNA species. When the aforementioned blot was probed using clone pRVF-34, which contains a DNA insert of approximately 2000 base of operations pairs representing a portion of virus M segment nearly the 3′ (Purchio et al., this volume), the resulting autoradiograph (fig. 4), illustrates that the middle band of the RNP RNA and the uppermost of the three bands in the pellet are homologous to sequences found in the M segment of the virus. The increased electrophoretic mobility of this band relative to the M segment of the genome suggests that this RNA is a subgenomic transcript and makes it a likely candidate for the glycoprotein messenger RNA.

FIGURE iv. Northern blot analysis of virus specific intracellular RNA. Infected Vero cells were fractionated on CsCl (8) and electrophoresed on agarose gels containing 8 mM methyl mercuric hydroxide. The RNA was then electroblotted to ABM paper (BioRad Laboratories). 32P-labeled RNP and pelleted RNA (lanes A and B), RNP and pelleted RNA probed with T1 fragments of virion RNA (lanes C and D); E and F, RNP and pelleted RNA probed with nick translated pRVF-34 (lanes Due east and F). Asterisk denotes two RNAs of nonviral origin which continued to be synthesized in the presence of Actinomycin D.

In club to determine the polypeptides encoded by the mRNAs in the pelleted RNA, total pelleted RNA was fractionated by preparative agarose gel electrophoresis. Because early experiments indicated that the mRNA for the North and NS polypeptides sedimented at approximately 12-18S on sucrose gradients, the portion of the gel encompassing RNA of this size class was fractionated, the RNA eluted and translated in a reticulocyte extract. In Effigy v, the open up arrow indicates the position of the South segment of vRNA in the agarose gel with fractions containing successively lower molecular weight RNA species to the correct. The information in Figure 5 signal that the maximum synthesis of N and NS polypeptides was directed by RNA in the molecular weight range of 300,000 daltons (lanes 6,vii,8). Information technology should exist noted that the maximum of translational activity for N and NS did not exactly coincide suggesting that at that place are separate messages for each polypeptide. In fact, ii bands of RNA in this region accept been occasionally resolved on denaturing agarose gels. The data indicate that the NS polypeptide was translated from an mRNA slightly larger than that for N protein. A 2d region of messenger activity coincided with the location of the RNA corresponding to the full size S genome segment (lane 1). Considering of the previous observation that the RNPs isolated from the cytoplasm contained positive stranded RNA, the RNA extracted from RNPs was also examined in an in vitro translation system. This RNA was likewise shown to yield N and NS polypeptides (lanes 11 and 12). Notwithstanding, while the relative amounts of the N and NS polypeptides synthesized in response to the 300,000 dalton mRNAs reflected the relative amounts of the two polypeptides synthesized in vivo (fig. iii) the yields of Due north and NS from the RNP RNA did not reverberate this same ratio.

FIGURE 5. In vitro translation of fractionated intracellular mRNA. The CsCl pelleted RNA was electrophoresed on 1.four% agarose gels containing 8 mM methyl mercuric hydroxide (7). The gel was fractionated equally described in the text, lanes 1–10. The pointer indicates the fraction containing the RNA which migrates as full length S segment. Translation products of RNA from intracellular RNP, lanes eleven, 0.5 μg RNA; lane 12, i μg RNA. Molecular weights of marking polypeptides (x 10−iii) are shown at the left.

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Nucleic acid quantification

Frank H. Stephenson , in Calculations for Molecular Biology and Biotechnology (Second Edition), 2010

five.7 Estimating DNA Concentration on an Ethidium Bromide-Stained Gel

Agarose gel electrophoresis is commonly used to separate DNA fragments following a restriction digest or PCR amplification. Fragments are detected past staining the gel with the intercalating dye, ethidium bromide, followed by visualization/photography nether UV light. Ethidium bromide stains Dna in a concentration-dependent manner such that the more than DNA that is present in a band on the gel, the more than intensely it will stain. This relationship makes information technology possible to estimate the quantity of Deoxyribonucleic acid present in a ring through comparison with another band of known DNA amount. If the intensities of 2 bands are like, then they contain like amounts of Dna. Ethidium bromide stains ssDNA and RNA only very poorly. These forms of nucleic acid will not give reliable quantitation by gel electrophoresis.

Problem 5.23

Five hundred nanograms (0.5   μg) of λ DNA digested with the brake endonuclease HindIII is loaded onto an agarose gel as a size marker. A ring generated from a DNA amplification experiment has the same intensity upon staining with ethidium bromide as the 564   bp fragment from the λ HindIII digest. What is the approximate amount of DNA in the amplified fragment?

Solution 5.23

This problem is solved past determining how much DNA is in the 564   bp fragment. Since the amplified Dna fragment has the same intensity later on staining equally the 564   bp fragment, the two bands comprise equivalent amounts of DNA.

Phage λ is 48   502   bp in length. The 564   bp HindIII fragment is to the total length of the phage λ genome as its amount (in ng) is to the full corporeality of λ HindIII mark run on the gel (500   ng). This allows the following relationship:

x ng 500 ng = 564 bp 48 502 bp

x ng = ( 500 ng ) ( 564 bp ) 48 502 bp = 5.8 ng

Therefore, there are approximately 5.8   ng of Dna in the band of the amplified DNA fragment.

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Detection of Alkali metal Phosphatase past Time-Resolved Fluorescence

Eva F. Gudgin Dickson , ... Alfred Poliak , in Nonisotopic Probing, Blotting, and Sequencing (2nd Edition), 1995

1 Dna Southern Blot Hybridization Analysis

The protocol for agarose gel electrophoresis and Southern transfer generally follows standard techniques. The loading buffer described below is recommended; the tracking dye should non be run in lanes containing the samples of interest, as the dye may interfere with uniform illumination of the samples during the concluding photography.

Solution Formulations
Loading buffer 15% Ficoll type 400 in deionized water
Denaturation solution 1.5 M NaCl, 0.5 Thousand NaOH
Neutralization solution one.0 Thousand Tris pH   viii.0, ane.v M NaCl
Transfer buffer 10 × SSC (0.15   Chiliad sodium citrate, 1.5   G NaCl, pH   seven.0)
6 × SSC 0.09 Grand sodium citrate, 0.ix Grand NaCl, pH   7.0
DNA dilution buffer Phosphate buffered saline (1.5   mM NaH2PO4, viii.0   mM 10002HPO4, 137   mM NaCl, two.five   m1000 KCl, pH   7.ii) containing 2   μg/ml sheared salmon sperm Deoxyribonucleic acid
twenty × SSC 0.iii M sodium citrate, 3.0 M NaCl, pH   7.0
Prehybridization buffer 6 X SSC, 50% formamide, 0.one% Ficoll, 0.i% polyvinylpyrrolidone, 0.five% powdered skim milk, 0.2   mg/ml freshly denatured sheared salmon sperm DNA, five% polyethyleneglycol
Hybridization solution 50–200   ng/ml of labeled DNA probe in prehybridization buffer
Membrane launder solution one 5 X SSC, 0.five% SDS
Membrane wash solution 2 0.one X SSC, 1% SDS
Membrane wash solution 3 two X SSC
TBS-T20 0.ane Grand Tris pH   7.v, 0.15 Chiliad NaCl, 0.05% Tween 20
Blocking solution 1% BSA in TBS-T20
Alkaline phosphatase-labeled streptavidin solution Working dilution of conjugate in TBS- T20, for case, ane:6000 dilution of ExtrAvidin streptavidin–alkaline phosphatase conjugate (Sigma), approx. 500   mU/ml
Substrate buffer 0.one M Tris pH   nine.0, 0.1 K NaCl, 1   1000M MgCl2
Substrate stock solution 10−   2M REALL-Chiliad in 0.one M NaOH
Substrate solution 10 × dilution of substrate stock solution in substrate buffer
Developing solution one × REALL Developing Reagent, 1 × REALL Developing Buffer in distilled, deionized water
1.

The transfer of the Deoxyribonucleic acid from the agarose gel to nylon membrane is performed as follows. Denature the Deoxyribonucleic acid by gently shaking the gel in dénaturation solution (two–3 gel volumes) for 30   min at room temperature; repeat this in one case. Neutralize the gel past gentle shaking in neutralization solution (2–3 gel volumes) for 30   min at room temperature. Check the pH of the gel with pH paper and echo neutralization pace if necessary. Perform the Southern transfer to nylon membrane cut to precisely the size of the gel and prewetted in transfer buffer. Perform the transfer in transfer buffer for 18   hr. Wash the membrane in 6X SSC for 5   min at room temperature, and allow it to dry for xxx   min on a sail of clean blotting newspaper. Irradiate the membrane with 254   nm UV light for 3   min, or alternately place in a vacuum oven at 80   °C for to 2   hour. The membrane tin be stored dry out at this betoken.

2.

Prehybridize the membrane in a sealed plastic bag for I to 2   hr at 42   °C in ten   ml prehybridization buffer. Remove the prehybridization buffer and add together 5   ml hybridization solution containing fifty–200   ng/ml biotinylated long probe. Seal the membrane in a plastic bag and hybridize at 42   °C overnight with shaking. Launder the membrane twice in 100   ml membrane wash solution I for 5   min at 65   °C, one time in 100   ml membrane wash solution 2 for 30   min at 65   °C (this wash solution temperature can be adjusted for desired level of stringency), and one time in 100   ml in membrane wash solution 3 for 5   min at room temperature.

3.

Soak the membrane for 5   min in 100   ml TBS-T20 and and so cake with 100   ml of blocking solution at 65   °C for I hr. Incubate the membrane with 50   ml of the element of group i phosphatase-labeled strep-tavidin solution for 10   min. Remove nonspecifically bound alkali metal phosphatase conjugate, past washing twice with 100   ml of TBS-T20 for 15   min and once with 100   ml substrate buffer for I hour.

iv.

Before adding the substrate solution, lay the membrane (DNA side upwards) on heavy blotting newspaper until the membrane is uniformly damp merely non moisture, to remove excess liquid. Place the membrane inside a development bag (consisting of a 0.4-mm thick transparent polyethylene plastic pocketbook that has been cut open on three sides) leaving a gap of about I cm around the edge of the membrane on all four sides. With the top of the purse pulled away, add one.0   ml of REALL-Thousand substrate solution in drops over the surface of the membrane. Close the tiptop of the bag gently over the surface of the membrane in lodge to exclude air bubbles and spread the solution. Using a 10   ml disposable pipet, coil over the top of the bag gently in several directions to ensure even distribution of the substrate. Incubate for I to 4   60 minutes in subdued lighting (longer incubations will reduce sharpness of bands without substantially increasing sensitivity). Do non handle the pocketbook during the incubation period, and at no time handle the membrane other than as described beneath, in guild to preclude smearing of the signal.

5.

After the desired incubation time has elapsed, turn the development bag containing the membrane face down and gently open up the back side of the bag to one side. Cut a piece of heavy blotting paper to a size larger than the membrane and apply information technology to the back side of the membrane. Remove excess substrate solution and and then remove the blotting paper. Add 1.5   ml of developing solution in drops to the dorsum of the membrane effectually all four sides. Shut the bag and gently gyre with a pipet. After a few seconds, absorb the excess solution from behind the membrane equally described above. At this indicate, seal the handbag to prevent leakage of luminescent solution and degradation of the luminescent signal. The membrane is now gear up for photography. Photograph the membrane inside two   hr of development. To photo the membrane in the TRP100, place the membrane in the plastic pocketbook in the sample tray of the TRP100 and clench in place, and so adjust height of the sample tray as needed to obtain correct focus. Select the right operating parameters for the TRP100 for use with REALL reagents. Photo the sample for an exposure time in the range of near 30   sec to three   min. Typical results of a Southern blotting analysis are presented in Fig. 4.

Fig. 4. Southern blot of BstN I assimilate of pBR322 DNA probed with biotinylated, nicktranslated pBR322 probe, REALL-M substrate was incubated for two   hr. Lanes contain (from left) 20, ten, 5, 2.5, 1.three, 0.63, 0.31   ng pBR322, and 543   ng Hind III assimilate λ-Deoxyribonucleic acid (not visible). Photographic exposure (a) twenty   sec; (b) twoscore   sec; (c) 80   sec.

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Expression of Fusion Protein from Positive Clones, SDS–PAGE and Western Blot: Part I

Susan Carson , ... Melissa C. Srougi , in Molecular Biology Techniques (4th Edition), 2019

I Introduction

SDS–Page is used to separate proteins by molecular weight. SDS–PAGE of proteins has numerous applications, including molecular weight decision, determining sample purity, quantifying expression, western blotting (immunoblotting), and isolating proteins for peptide sequencing or for generating antibodies.

You are already familiar with DNA agarose gel electrophoresis, and SDS–PAGE shares some similarities with this method. Both methods separate molecules by size, use electrical accuse differences to cause migration and both require a matrix to carve up molecules by size. However, the structural and biochemical differences between DNA and proteins pb to a number of variations in their separation by electrophoresis.

1.

Proteins are generally smaller than DNA. Thus, while DNA (larger than 100   bp) is routinely separated on agarose gels, proteins are more often than not run on polyacrylamide gels, as polyacrylamide matrices have a smaller pore (sieve) size than agarose. DNA fragments smaller than 100   bp are often separated using polyacrylamide.

2.

All Deoxyribonucleic acid is negatively charged, but proteins have varying charges depending on the amino acid content of the specific polypeptide and the pH of the buffer. Some proteins are positively charged, while some carry a cyberspace negative charge.

three.

Dna, peculiarly linear Dna, has little secondary structure, while proteins tin be globular or linear and have quaternary structure, such equally dimers and other multimers.

Because of numbers ii and iii, if proteins were run on a native or not-denaturing polyacrylamide gel (i.east., run without SDS), protein migration would depend on at to the lowest degree three factors: size, accuse, and shape.

SDS–Page allows proteins to drift past size alone, through the utilise of SDS and a reducing agent. SDS is an ionic detergent that denatures (unfolds) proteins past wrapping effectually the polypeptide backbone forming a micelle, and thus conferring a net negative charge in proportion to polypeptide length. SDS also disrupts most non-covalent interactions, such as electrostatic interactions and hydrogen bonds, thereby decreasing protein folding. A reducing agent such equally β-mercaptoethanol or dithiothreitol is added to reduce disulfide bonds (cystine bonds) and further unfold the proteins.

After boiling a protein sample in SDS and β-mercaptoethanol, proteins act as negatively charged linear molecules and can be electrophoretically separated past size lone (Fig. 9.1).

Figure ix.1. Polypeptide treated with β-mercaptoethanol and SDS. β-Mercaptoethanol breaks the covalent disulfide (cysteine) bonds. SDS disrupts non-covalent interactions, farther denaturing the protein, and forms a micelle around it, conferring a negative accuse. SDS, Sodium dodecyl sulfate.

Subsequently running the gel, it can either exist stained non-specifically to visualize the protein bands using Coomassie Blue, GelCode Blue, or silvery stain; or the proteins can be transferred to a nitrocellulose membrane for western blotting (immunoblotting) to visualize a specific protein of involvement. In Lab Session 12, Analysis of Purification Fractions, we will run an SDS–PAGE gel and stain it using GelCode Blue to visualize protein bands.

In today's lab session, nosotros will begin a western absorb (to exist completed in the following laboratory session). The outset footstep of this process is to set the protein samples and separate them using SDS–PAGE. Then, the proteins from the polyacrylamide gel are transferred to the nitrocellulose membrane. Full protein on the nitrocellulose membrane may be visualized at this bespeak using the h2o-soluble Ponceau stain. After the proteins are transferred, a monoclonal antibody confronting GFP is used to specifically visualize your GST::EGFP fusion protein (more data on this in Lab Session x: Expression of Fusion Protein from Positive Clones, SDS–Page, and Western Blot: Part II). This portion of the western blot will be completed in the side by side laboratory session.

The molecular weight of the GST::EGFP fusion protein tin can exist estimated, assuming the boilerplate weight per amino acid is equal to 114   Da. The gst cistron is 660   bp, encoding 220 amino acids: 220×114=25,080   Da. The egfp gene is 720   bp, encoding 240 amino acids: 240×114=27,360   Da. In that location are 174 additional nucleotides between gst and egfp, encoding 58 amino acids: 58×114=6612   Da. The weight of the fusion poly peptide tin can therefore be approximated equally: 25,080+27,360+6612=59,052   Da or ~59   kDa. Yous can determine the bodily molecular weight (using the molecular weight for each amino acid) using free online software; the verbal molecular weight of the GST::EGFP fusion protein is 58,500   Da. You will exist able to non-specifically visualize a poly peptide ring of this approximate size in your positive clones using the Ponceau stain. The completion of the western blot practise side by side week will use an antibiotic specific for EGFP to confirm that the band is indeed GST::EGFP. In the negative clones, after Ponceau staining, you lot may see a ring of approximately 25   kDa, corresponding to the GST protein alone.

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λSV2, a Plasmid Cloning Vector that Can Be Stably Integrated in Escherichia coli

BRUCE H. HOWARD , MAX Due east. GOTTESMAN , in Experimental Manipulation of Gene Expression, 1983

C. Construction of Recombinant Plasmids

Deoxyribonucleic acid restriction fragments were separated past agarose-gel electrophoresis in 0.04 M Tris acetate and 0.002 Grand EDTA (pH viii.one) containing 10 μgm/ml ethidium bromide, visualized by longwave UV illumination (Ultraviolet Products, San Gabriel, California), and eluted from excised gel slices every bit described by Chen and Thomas (1980). The v′ recessed restriction-fragment ends were converted to "blunt" ends by incubation with Deoxyribonucleic acid polymerase I (Seeburg et al., 1977); 3′ recessed brake-fragment ends were converted to blunt ends by incubation with AMV contrary transcriptase (ane unit of measurement/nmol fragment ends) for 30 min at 37°C. Purified restriction fragments were joined by incubation with T4 DNA ligase overnight at 14°C. Bacterial transformations of E. coli strain HB101 were carried out by the CaClii method (Mandel and Higa, 1970). For transformation of E. coli strain N6106, leaner were grown in LB broth supplemented with 0.003% biotin and shifted between 32 and 42°C as described in Section III. Transformants were selected for growth in agar containing l μgm/ml ampicillin or 15 μgm/ml chloramphenicol.

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